Device and method for pressure-driven plug transport and reaction

ABSTRACT

The present invention provides microfabricated substrates and methods of conducting reactions within these substrates. The reactions occur in plugs transported in the flow of a carrier-fluid.

This application is a continuation of U.S. application Ser. No.14,507,938, filed on Oct. 7, 2014, which is a continuation of U.S.application Ser. No. 11/589,700, filed on Oct. 30, 2006 (issued as U.S.Pat. No. 8,889,083), which is a divisional of U.S. application Ser. No.10/434,970, filed on May 9, 2003 (issued as U.S. Pat. No. 7,129,091),which claimed priority to U.S. Provisional Application No. 60/379,927,filed on May 9, 2002 and which also claimed priority to U.S. ProvisionalApplication No. 60/394,544 filed on Jul. 8, 2002, the contents of eachof which are incorporated herein by reference in their entirety.

BACKGROUND

Nonlinear dynamics, in conjunction with microfluidics, play a centralrole in the design of the devices and the methods according to theinvention. Microfluidics deals with the transport of fluids throughnetworks of channels, typically having micrometer dimensions.Microfluidic systems (sometimes called labs-on-a-chip) find applicationsin microscale chemical and biological analysis (micro-total-analysissystems). The main advantages of microfluidic systems are high speed andlow consumption of reagents. They are thus very promising for medicaldiagnostics and high-throughput screening. Highly parallel arrays ofmicrofluidic systems are used for the synthesis of macroscopicquantities of chemical and biological compounds, e.g., the destructionof chemical warfare agents and pharmaceuticals synthesis. Theiradvantage is improved control over mass and heat transport.

Microfluidic systems generally require means of pumping fluids throughthe channels. In the two most common methods, the fluids are eitherdriven by pressure or driven by electroosmotic flow (EOF). Flows drivenby EOF are attractive because they can be easily controlled even incomplicated networks. EOF-driven flows have flat, plug-like velocityprofile, that is, the velocity of the fluid is the same near the wallsand in the middle of the channel. Thus, if small volumes of multipleanalytes are injected sequentially into a channel, these plugs aretransported as non-overlapping plugs (low dispersion), in which case thedispersion comes mostly from the diffusion between plugs. A maindisadvantage of EOF is that it is generated by the motion of the doublelayer at the charged surfaces of the channel walls. EOF can therefore behighly sensitive to surface contamination by charged impurities. Thismay not be an issue when using channels with negative surface charges inDNA analysis and manipulation because DNA is uniformly negativelycharged and does not adsorb to the walls. However, this can be a seriouslimitation in applications that involve proteins that are often chargedand tend to adsorb on charged surfaces. In addition, high voltages areoften undesirable, or sources of high voltages such as portableanalyzers may not be available.

Flows driven by pressure are typically significantly less sensitive tosurface chemistry than EOF. The main disadvantage of pressure-drivenflows is that they normally have a parabolic flow profile instead of theflat profile of EOF. Solutes in the middle of the channel move muchfaster (about twice the average velocity of the flow) than solutes nearthe walls of the channels. A parabolic velocity profile normally leadsto high dispersion in pressure-driven flows; a plug of solute injectedinto a channel is immediately distorted and stretched along the channel.This distortion is somewhat reduced by solute transport via diffusionfrom the middle of the channel towards the walls and back. But thedistortion is made worse by diffusion along the channel (the overalldispersion is known as Taylor dispersion).

Taylor dispersion broadens and dilutes sample plugs. Some of the sampleis frequently left behind the plug as a tail. Overlap of these tailsusually leads to cross-contamination of samples in different plugs.Thus, samples are often introduced into the channels individually,separated by buffer washes. On the other hand, interleaving samples withlong buffer plugs, or washing the system with buffer between samples,reduces the throughput of the system.

In EOF, flow transport is essentially linear, that is, if two reactantsare introduced into a plug and transported by EOF, their residence time(and reaction time) can be calculated simply by dividing the distancetraveled in the channel by the velocity. This linear transport allowsprecise control of residence times through a proper adjustment of thechannel lengths and flow rates. In contrast, dispersion inpressure-driven flow typically creates a broad range of residence timesfor a plug traveling in such flows, and this diminishes time control.

The issue of time control is important. Many chemical and biochemicalprocesses occur on particular time scales, and measurement of reactiontimes can be indicative of concentrations of reagents or theirreactivity. Stopped-flow type instruments are typically used to performthese measurements. These instruments rely on turbulent flow to mix thereagents and transport them with minimal dispersion. Turbulent flownormally occurs in tubes with large diameter and at high flow rates.Thus stopped-flow instruments tend to use large volumes of reagents (onthe order of ml/s). A microfluidic analog of stopped-flow, whichconsumes smaller volumes of reagents (typically μL/min), could be usefulas a scientific instrument, e.g., as a diagnostic instrument. So far,microfluidic devices have not be able to compete with stopped-flow typeinstruments because EOF is usually very slow (although with lessdispersion) while pressure-driven flows suffer from dispersion.

In addition, mixing in microfluidic systems is often slow regardless ofthe method used to drive the fluid because flow is laminar in thesesystems (as opposed to turbulent in larger systems). Mixing in laminarflows relies on diffusion and is especially slow for larger moleculessuch as DNA and proteins.

In addition, particulates present handling difficulty in microfluidicsystems. While suspensions of cells in aqueous buffers can be relativelyeasy to handle because cells are isodense with these buffers,particulates that are not isodense with the fluid tend to settle at thebottom of the channel, thus eventually blocking the channel. Therefore,samples for analysis often require filtration to remove particulates.

SUMMARY ACCORDING TO THE INVENTION

In accordance with the invention, a method of conducting a reactionwithin a substrate is provided that comprises introducing acarrier-fluid into a first channel of the substrate; introducing atleast two different plug-fluids into the first channel; and applyingpressure to the first channel to induce a fluid flow in the substrate toform substantially identical plugs comprising a mixture of plug-fluids.The plug-fluids are immiscible with the carrier-fluid. During plugformation, the cross-section of the plug is substantially similar to thecross-section of the first channel, so that the plug is substantially incontact with all walls of the first channel. After plug formation, thecross-section of the plug may be smaller than the cross-section of thechannel. A thin layer of carrier-fluid typically exists between the wallof the channel and the plug, although in some cases this layerdisappears. In general, each plug is substantially similar in size wheninitially formed in the channel. In addition, the capillary number ofthe plug in the channel is low, typically less than 1, preferably ≤about0.2, more preferably ≤about 0.1.

When plugs are formed from more than one plug-fluid, the fluids arerapidly mixed. Mixing inside plugs is further enhanced when the channelsare not straight (i.e., when chaotic flows are generated). Aperiodicchannel designs are preferred to induce rapid mixing within plugs. Inother cases, mixing can be slowed down or controlled such as by usingwinding channels, varying the fluid viscosities, varying the plug-fluidcomposition, and twirling, which can also be controlled.

The device of the present invention can be used to merge one or moreplug fluids. The plug-fluids are introduced either through a singleinlet or from multiple inlets. When the plug-fluids are introducedthrough a single inlet, they are preferably mixed just upstream of theinlet, so that substantial mixing does not occur prior to introductioninto the first channel. When plugs fluids are introduced throughmultiple inlets, one or more physical properties (such as the viscosity,plug dimensions, surface tension at the interface between the plugfluids and the carrier-fluid, or the surface tension at the interfacebetween the plug fluids and the walls of the channel) of the plug-fluidsare adjusted so that plugs composed of different plug-fluids merge intoa series of plugs prior to the outlet (that is, a series of plugs areformed which are composed of a mixture of plug-fluids). Alternatively,the plug-fluids can be introduced into separate channels to form plugscomposed of single plug-fluids. These channels are then merged into asingle merged channel. The continuous fluid flow within the substrateforms merged plugs in the single merged channel.

The device of the present invention can be used to split plugs into twoor more channels. Using the above devices and techniques, a variety ofreactions can be conducted, including polymerizations, crystallizations(including small molecule and proteins), nanoparticle synthesis,formation of unstable intermediates, enzyme-catalyzed reactions andassays, protein-protein binding, etc. More than one reaction can beconducted, either simultaneously or sequentially.

Further, the present invention also provides a device comprising one ormore substrates in accordance with the present invention.

The devices and methods according to the invention include variousnon-limiting embodiments or modifications several of which are discussedin details below.

BRIEF DESCRIPTION OF THE DRAWINGS AND PHOTOGRAPHS

FIG. 1A is a schematic diagram of a basic channel design that may beused to induce rapid mixing in plugs. FIG. 1B(1)-(4) are schematicdiagrams depicting a series of periodic variations of the basic channeldesign. FIG. 1C(1)-(4) are schematic diagrams depicting a series ofaperiodic combinations resulting from a sequence of alternating elementstaken from a basic design element shown in FIG. 1A and an element fromthe periodic variation series shown in FIGS. 1B(1)-(4).

FIGS. 2A(1) and 2A(2) are schematic diagrams contrasting laminar flowtransport and plug transport in a channel, respectively. FIG. 2B(1)shows a photograph (right side, top portion) illustrating rapid mixinginside plugs moving through winding channels. FIG. 2B(2) shows aphotograph (right side, lower portion) showing that winding channels donot accelerate mixing in a laminar flow in the absence of PFD.

FIGS. 3A and 3B show photographs (right side) and schematic diagrams(left side) that depict a stream of plugs from an aqueous plug-fluid andan oil (carrier-fluid) in curved channels at flow rates of 0.5 μL/min(FIG. 3A) and 1.0 μL/min (FIG. 3B).

FIG. 4 shows a photograph (lower portion) and a schematic diagram (upperportion) that illustrate plug formation through the injection of oil andmultiple plug-fluids.

FIG. 5 is a schematic diagram that illustrates a two-step reaction inwhich plugs are formed through the injection of oil and multipleplug-fluids using a combination of different geometries for controllingreactions and mixing.

FIG. 6 is a schematic representation of part of a microfluidic networkthat uses multiple inlets and that allows for both splitting and mergingof plugs. This schematic diagram shows two reactions that are conductedsimultaneously. A third reaction (between the first two reactionmixtures) is conducted using precise time delay.

FIGS. 7(a)-(b) show microphotographs (10 μs exposure) illustrating rapidmixing inside plugs (a) and negligible mixing in a laminar flow (b)moving through winding channels at the same total flow velocity. FIG.7(c) shows a false-color microphotograph (2 s exposure, individual plugsare invisible) showing time-averaged fluorescence arising from rapidmixing inside plugs of solutions of Fluo-4 and CaCl₂. FIGS. 7(d)(i) and7(d)(ii) show plots of the relative normalized intensity (I) offluorescence obtained from images such as shown in (c) as a function ofdistance (FIG. 7(d)(i)) traveled by the plugs and of time required totravel that distance (FIG. 7(d)(ii)) at a given flow rate. FIG. 7(e)shows a false-color microphotograph (2 s exposure) of the weakfluorescence arising from negligible mixing in a laminar flow of thesolutions used in (c).

FIGS. 8(a)-8(d) show photographs (FIGS. 8(b) and (d)) and schematics(FIGS. 8(a) and (c)) that illustrate fast mixing at flow rates of about0.5 μL/min (FIGS. 8(a) and (b)) and about 1.0 μL/min (FIGS. 8(c) and(d)) using 90°-step channels.

FIGS. 9(a)-9(d) show schematics (FIGS. 9(a) and 9(c)) and photographs(FIGS. 9(b) and 9(d)) illustrating fast mixing at flow rates of about1.0 μL/min (FIGS. 9(a) and 9(b)) and about 0.5 μL/min (FIGS. 9(c) and9(d)) using 135°-step channels.

FIG. 10(a) is a schematic diagram depicting three-dimensional confocalvisualization of chaotic flows in plugs. FIG. 10(b) is a plot showing asequence preferably used for visualization of a three-dimensional flow.

FIG. 11 shows a schematic diagram of a channel geometry designed toimplement and visualize the baker's transformation of plugs flowingthrough microfluidic channels.

FIGS. 12(a) and 12(b) show photographs depicting the merging of plugs(FIG. 12(a)) and splitting of plugs (FIG. 12(b)) that flow in separatechannels or channel branches that are perpendicular.

FIG. 13 shows UV-VIS spectra of CdS nanoparticles formed by rapid mixingin plugs (spectrum with a sharp absorption peak) and by conventionalmixing of solutions

FIGS. 14(a)(i)-(a)(iii) and 14(b)(i)-(b)(iii) show schematic diagrams(FIGS. 14(a)(i) and 14(b)(i)) and photographs (FIGS. 14(a)(ii),14(a)(iii), 14(b)(ii) and 14(b)(iii)) that illustrate the synthesis ofCdS nanoparticles in PDMS microfluidic channels in single-phase aqueouslaminar flow (FIG. 14(a)(i)-(iii)) and in aqueous plugs that aresurrounded by water-immiscible perfluorodecaline (FIG. 14(b)(i)-(iii))

FIG. 15 shows schematic representations of the synthesis of CdSnanoparticles inside plugs

FIG. 16 is a schematic illustration of a microfluidic device accordingto the invention that illustrates the trapping of plugs

FIG. 17 is a schematic of a microfluidic method for forming plugs withvariable compositions for protein crystallization

FIGS. 18 (a) and (b) are schematic illustrations of a method forcontrolling heterogeneous nucleation by varying the surface chemistry atthe interface of an aqueous plug-fluid and a carrier-fluid.

FIG. 19 is a schematic diagram that illustrates a method of separatingnucleation and growth using a microfluidic network according to thepresent invention.

FIGS. 20 (a) and (b) show schematic diagrams that illustrate two methodsthat provide a precise and reproducible degree of control over mixingand that can be used to determine the effect of mixing on proteincrystallization.

FIG. 21 is a reaction diagram illustrating an unstable point in thechlorite-thiosulfate reaction.

FIGS. 22A-22D are schematic diagrams that show various examples ofgeometries of microfluidic channels according to the invention forobtaining kinetic information from single optical images.

FIG. 23 shows a schematic of a microfluidic network (left side) and atable of parameters for a network having channel heights of 15 and 2 μm.

FIGS. 24(a)-24(c) show a reaction scheme that depicts examples offluorinated surfactants that form monolayers that are: (a) resistant toprotein adsorption; (b) positively charged; and (c) negatively charged.FIG. 24b shows a chemical structure of neutral surfactants charged byinteractions with water by protonation of an amine or a guanidiniumgroup. FIG. 24c shows a chemical structure of neutral surfactantscharged by interactions with water deprotonation of a carboxylic acidgroup.

FIGS. 25(a)-25(c) are schematic diagrams of a microfluidic network (leftside of 25(a), 25(b), and 25(c)) that can be used for controlling theconcentrations of aqueous solutions inside the plugs, as well asphotographs (right side of 25(a), 25(b), and 25(c)) showing theformation of plugs with different concentrations of the aqueous streams.

FIGS. 26(a) and 26(b) are schematic diagrams of microfluidic network(left side of 26(a) and 26(b)) and photographs (right side of 26(a) and26(b)) of the plug-forming region of the network in which the aqueousstreams were dyed with red and green food dyes to show their flowpatterns.

FIGS. 27(a)(1)-(a)(2), 27(b), and 27(c)(1)-(c)(2) are schematics,photographs and plots showing the effects of initial conditions onmixing by recirculating flow inside plugs moving through straightmicrochannels. FIG. 27(a)(1) is a schematic diagram showing thatrecirculating flow (shown by black arrows) efficiently mixed solutionsof reagents that were initially localized in the front and back halvesof the plug. FIG. 27(a)(2) is a schematic diagram showing thatrecirculating flow (shown by black arrows) did not efficiently mixsolutions of reagents that were initially localized in the left andright halves of the plugs

FIG. 27(b) shows a schematic diagram showing the inlet portions (leftside) and photographs of images showing measurements of various periodsand lengths of plugs. FIG. 27(c) 1) shows a graph of the relativeoptical intensity of Fe(SCN)_(x) ^((3−x)+) complexes in plugs of varyinglengths. FIG. 27(c) 2) is the same as FIG. 27c 1) except that each plugtraverses a distance of 1.3 mm.

FIG. 28 is a schematic illustration of a plug showing the notation usedto identify different regions of the plugs relative to the direction ofmotion.

FIGS. 29(a)-b) are plots of the periods and the lengths of plugs as afunction of total flow velocity (FIG. 29(a)) and water fraction (FIG.29(b)).

FIG. 30 shows photographs illustrating weak dependence of periods,length of plugs, and flow patterns inside plugs on total flow velocity.

FIGS. 31A-31C are plots showing the distribution of periods and lengthsof plugs where the water fractions were 0.20, 0.40, and 0.73,respectively.

FIG. 32 shows photographs (middle and right side) that show that plugtraps are not required for crystal formation in a microfluidic network,as well as a diagram of the microfluidic network (left side).

FIGS. 33a-d (left side) are top views of microfluidic networks (leftside) and photographs (right side) that comprise channels having eitheruniform or nonuniform dimension. FIG. 33a shows that merging of theplugs occurs infrequently in the T-shaped channel shown in thephotographs. FIG. 33b illustrates plug merging occurring between plugsarriving at different times at the Y-shaped junction (magnified viewshown). FIG. 33c depicts in-phase merging, i.e., plug merging uponsimultaneous arrival of at least two plugs at a junction, of plugs ofdifferent sizes generated using different oil/water ratios at the twopairs of inlets. FIG. 33d illustrates defects (i.e., plugs that fail toundergo merging when they would normally merge under typical or idealconditions) produced by fluctuations in the relative velocity of the twoincoming streams of plugs.

FIGS. 34a-c show a schematic diagram (a, left side) and photographs (b,c) each of which depicts a channel network viewed from the top. FIG. 34ais a schematic diagram of the channel network used in the experiment.FIG. 34b is a photograph showing the splitting of plugs into plugs ofapproximately one-half the size of the initial plugs. FIG. 34c is aphotograph showing the asymmetric splitting of plugs which occurred whenP₁<P₂.

FIG. 35 shows a schematic diagram (a, left side) and photographs (b, c)that depicts the splitting of plugs using microfluidic networks withoutconstrictions near the junction.

FIG. 36 shows a photograph (right side) of lysozyme crystals grown inwater plugs in the wells of the microfluidic channel, as well as adiagram (left side) of the microfluidic network used in thecrystallization.

FIG. 37 is a schematic diagram that depicts a microfluidic deviceaccording to the invention that can be used to amplify a small chemicalsignal using an autocatalytic (and possibly unstable) reaction mixture.

FIG. 38 is a schematic diagram that illustrates a method for amulti-stage chemical amplification which can be used to detect as few asa single molecule.

FIG. 39 shows a diagram (left side) of the microfluidic network and aphotograph (right side) of water plugs attached to the PDMS wall.

FIG. 40A is a schematic representation (left side) of a microfluidicnetwork used to measure kinetics data for the reaction of RNase A usinga fluorogenic substrate (on-chip enzyme kinetics), and FIG. 40 and FIG.40B are plots that shows the kinetic data for the reaction between RNaseA and a fluorogenic substrate.

FIGS. 41(a)-41(c) show a photograph (FIGS. 41(b) and 41(c), middle andright side) of the water droplet region of the microfluidic network (Tstands for time), as well as a diagram of the microfluidic network (FIG.41(a), left side).

FIG. 42 shows a schematic diagram (left side) of a microfluidic networkand a photograph (right side) of the ink plug region of the microfluidicnetwork in which the gradients were formed by varying the flow rates.

FIG. 43 shows a schematic diagram (left side) of a microfluidic networkand a photograph (right side) of lysozyme crystals formed in themicrofluidic network using gradients.

FIGS. 44(a)-44(d) are schematic illustrations showing how an initialgradient may be created by injecting a discrete aqueous sample of areagent B into a flowing stream of water.

FIG. 45(a) shows a schematic of the microfluidic network used todemonstrate that on-chip dilutions can be accomplished by varying theflow rates of the reagents. The blue rectangle outlines the field ofview for images shown in FIG. 45(c)-(d). FIG. 45(b) shows a graphquantifying this dilution method by measuring fluorescence of a solutionof fluorescein diluted in plugs in the microchannel.

DETAILED DESCRIPTION ACCORDING TO THE INVENTION

The term “analysis” generally refers to a process or step involvingphysical, chemical, biochemical, or biological analysis that includescharacterization, testing, measurement, optimization, separation,synthesis, addition, filtration, dissolution, or mixing.

The term “analysis unit” refers to a part of or a location in asubstrate or channel wherein a chemical undergoes one or more types ofanalyses.

The term “carrier-fluid” refers to a fluid that is immiscible with aplug-fluid. The carrier-fluid may comprise a substance having both polarand non-polar groups or moieties.

The term “channel” refers to a conduit that is typically enclosed,although it may be at least partially open, and that allows the passagethrough it of one or more types of substances or mixtures, which may behomogeneous or heterogeneous, including compounds, solvents, solutions,emulsions, or dispersions, any one of which may be in the solid, liquid,or gaseous phase. A channel can assume any form or shape such as tubularor cylindrical, a uniform or variable (e.g., tapered) diameter along itslength, and one or more cross-sectional shapes along its length such asrectangular, circular, or triangular. A channel is typically made of asuitable material such as a polymer, metal, glass, composite, or otherrelatively inert materials. As used herein, the term “channel” includesmicrochannels that are of dimensions suitable for use in devices. Anetwork of channels refers to a multiplicity of channels that aretypically connected or in communication with each other. A channel maybe connected to at least one other channel through another type ofconduit such as a valve.

The term “chemical” refers to a substance, compound, mixture, solution,emulsion, dispersion, molecule, ion, dimer, macromolecule such as apolymer or protein, biomolecule, precipitate, crystal, chemical moietyor group, particle, nanoparticle, reagent, reaction product, solvent, orfluid any one of which may exist in the solid, liquid, or gaseous state,and which is typically the subject of an analysis.

The term “detection region” refers to a part of or a location in asubstrate or channel wherein a chemical is identified, measured, orsorted based on a predetermined property or characteristic.

The term “device” refers to a device fabricated or manufactured usingtechniques such as wet or dry etching and/or conventional lithographictechniques or a micromachining technology such as soft lithography. Asused herein, the term “devices” includes those that are called, known,or classified as microfabricated devices. A device according to theinvention may have dimensions between about 0.3 cm to about 15 (for 6inch wafer) cm per side and between about 1 micrometer to about 1 cmthick, but the dimensions of the device may also lie outside theseranges.

The term “discrimination region” refers to a part of or a location in asubstrate or channel wherein the flow of a fluid can change direction toenter at least one other channel such as a branch channel.

The term “downstream” refers to a position relative to an initialposition which is reached after the fluid flows past the initial point.In a circulating flow device, downstream refers to a position fartheralong the flow path of the fluid before it crosses the initial pointagain. “Upstream” refers to a point in the flow path of a fluid that thefluid reaches or passes before it reaches or passes a given initialpoint in a substrate or device.

The term “flow” means any movement of a solid or a fluid such as aliquid. For example, the movement of plug-fluid, carrier-fluid, or aplug in a substrate, or component of a substrate according to theinvention, or in a substrate or component of a substrate involving amethod according to the invention, e.g., through channels of amicrofluidic substrate according to the invention, comprises a flow. Theapplication of any force may be used to provide a flow, includingwithout limitation: pressure, capillary action, electro-osmosis,electrophoresis, dielectrophoresis, optical tweezers, and combinationsthereof, without regard for any particular theory or mechanism ofaction.

The term “immiscible” refers to the resistance to mixing of at least twophases or fluids under a given condition or set of conditions (e.g.,temperature and/or pressure) such that the at least two phases or fluidspersist or remain at least partially separated even after the phaseshave undergone some type of mechanical or physical agitation. Phases orfluids that are immiscible are typically physically and/or chemicallydiscernible, or they may be separated at least to a certain extent.

The term “inlet port” refers to an area of a substrate that receivesplug-fluids. The inlet port may contain an inlet channel, a well orreservoir, an opening, and other features that facilitate the entry ofchemicals into the substrate. A substrate may contain more than oneinlet port if desired. The inlet port can be in fluid communication witha channel or separated from the channel by a valve.

The term “nanoparticles” refers to atomic, molecular or macromolecularparticles typically in the length scale of approximately 1-100 nanometerrange. Typically, the novel and differentiating properties and functionsof nanoparticles are observed or developed at a critical length scale ofmatter typically under 100 nm. Nanoparticles may be used in constructingnanoscale structures and they may be integrated into larger materialcomponents, systems and architectures. In some particular cases, thecritical length scale for novel properties and phenomena involvingnanoparticles may be under 1 nm (e.g., manipulation of atoms atapproximately 0.1 nm) or it may be larger than 100 nm (e.g.,nanoparticle reinforced polymers have the unique feature atapproximately 200-300 nm as a function of the local bridges or bondsbetween the nanoparticles and the polymer).

The term “nucleation composition” refers to a substance or mixture thatincludes one or more nuclei capable of growing into a crystal underconditions suitable for crystal formation. A nucleation composition may,for example, be induced to undergo crystallization by evaporation,changes in reagent concentration, adding a substance such as aprecipitant, seeding with a solid material, mechanical agitation, orscratching of a surface in contact with the nucleation composition.

The term “outlet port” refers to an area of a substrate that collects ordispenses the plug-fluid, carrier-fluid, plugs or reaction product. Asubstrate may contain more than one outlet port if desired.

The term “particles” means any discrete form or unit of matter. The term“particle” or “particles” includes atoms, molecules, ions, dimers,polymers, or biomolecules.

The term “particulate” refers to a cluster or agglomeration of particlessuch as atoms, molecules, ions, dimers, polymers, or biomolecules.Particulates may comprise solid matter or be substantially solid, butthey may also be porous or partially hollow. They may contain a liquidor gas. In addition, particulates may be homogeneous or heterogeneous,that is, they may comprise one or more substances or materials.

“Plugs” in accordance with the present invention are formed in asubstrate when a stream of at least one plug-fluid is introduced intothe flow of a carrier-fluid in which it is substantially immiscible. Theflow of the fluids in the device is induced by a driving force orstimulus that arises, directly or indirectly, from the presence orapplication of, for example, pressure, radiation, heat, vibration, soundwaves, an electric field, or a magnetic field. Plugs in accordance withthe present invention may vary in size but when formed, theircross-section should be substantially similar to the cross-section ofthe channels in which they are formed. When plugs merge or get trappedinside plug traps, the cross-section of the plugs may change. Forexample, when a plug enters a wider channel, its cross-section typicallyincreases.

Further, plugs in accordance with the present invention may vary inshape, and for example may be spherical or non-spherical. The shape ofthe plug may be independent of the shape of the channel (e.g., a plugmay be a deformed sphere traveling in a rectangular channel). The plugsmay be in the form of plugs comprising an aqueous plug-fluid containingone or more reagents and/or one or more products formed from a reactionof the reagents, wherein the aqueous plug-fluid is surrounded by anon-polar or hydrophobic fluid such as an oil. The plugs may also be inthe form of plugs comprising mainly a non-polar or hydrophobic fluidwhich is surrounded by an aqueous fluid. The plugs may be encased by oneor more layers of molecules that comprise both hydrophobic andhydrophilic groups or moieties. The term “plugs” also includes plugscomprising one or more smaller plugs, that is, plugs-within-plugs. Therelative amounts of reagents and reaction products contained in theplugs at any given time depend on factors such as the extent of areaction occurring within the plugs. Preferably, plugs contain a mixtureof at least two plug fluids.

The term “plug-forming region” refers to a junction between an inletport and the first channel of a substrate according to the invention.Preferably, the fluid introduced into the inlet port is “incompatible”(i.e., immiscible) with the fluid in the first channel so that plugs ofthe fluid formed in the plug-forming region are entrained into thestream of fluid from the first channel.

The term “plug-fluid” refers to a fluid wherein or using which areaction or precipitation can occur. Typically, the plug-fluid containsa solvent and a reagent although in some embodiments at least oneplug-fluid may not contain a reagent. The reagent may be soluble orinsoluble in the solvent. The plug-fluid may contain a surfactant. Atleast two different plug-fluids are used in the present invention. Whenboth plug-fluids contain reagents, the fluids are typically miscible,but can also be partially immiscible, so long as the reagents withineach plug-fluid can react to form at least one product or intermediate.

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together. Polymers include both condensation andaddition polymers. Typical examples of condensation polymers includepolyamide, polyester, protein, wool, silk, polyurethane, cellulose, andpolysiloxane. Examples of addition polymers are polyethylene,polyisobutylene, polyacrylonitrile, poly(vinyl chloride), andpolystyrene. Other examples include polymers having enhanced electricalor optical properties (e.g., a nonlinear optical property) such aselectroconductive or photorefractive polymers. Polymers include bothlinear and branched polymers.

The term “protein” generally refers to a set of amino acids linkedtogether usually in a specific sequence. A protein can be eithernaturally-occurring or man-made. As used herein, the term “protein”includes amino acid sequences that have been modified to containmoieties or groups such as sugars, polymers, metalloorganic groups,fluorescent or light-emitting groups, moieties or groups that enhance orparticipate in a process such as intramolecular or intermolecularelectron transfer, moieties or groups that facilitate or induce aprotein into assuming a particular conformation or series ofconformations, moieties or groups that hinder or inhibit a protein fromassuming a particular conformation or series of conformations, moietiesor groups that induce, enhance, or inhibit protein folding, or othermoieties or groups that are incorporated into the amino acid sequenceand that are intended to modify the sequence's chemical, biochemical, orbiological properties. As used herein, a protein includes, but is notlimited to, enzymes, structural elements, antibodies, hormones, electroncarriers, and other macromolecules that are involved in processes suchas cellular processes or activities. Proteins typically have up to fourstructural levels that include primary, secondary, tertiary, andquaternary structures.

The term “reaction” refers to a physical, chemical, biochemical, orbiological transformation that involves at least one chemical, e.g.,reactant, reagent, phase, carrier-fluid, or plug-fluid and thatgenerally involves (in the case of chemical, biochemical, and biologicaltransformations) the breaking or formation of one or more bonds such ascovalent, noncovalent, van der Waals, hydrogen, or ionic bonds. The termincludes typical chemical reactions such as synthesis reactions,neutralization reactions, decomposition reactions, displacementreactions, reduction-oxidation reactions, precipitation,crystallization, combustion reactions, and polymerization reactions, aswell as covalent and noncovalent binding, phase change, color change,phase formation, crystallization, dissolution, light emission, changesof light absorption or emissive properties, temperature change or heatabsorption or emission, conformational change, and folding or unfoldingof a macromolecule such as a protein.

The term “reagent” refers to a component of a plug-fluid that undergoesor participates (e.g., by influencing the rate of a reaction or positionof equilibrium) in at least one type of reaction with one or morecomponents of other plug-fluids or a reagent-containing carrier-fluid inthe substrate to produce one or more reaction products or intermediateswhich may undergo a further reaction or series of reactions.

A reagent contained in a plug-fluid may undergo a reaction in which astimulus such as radiation, heat, temperature or pressure change,ultrasonic wave, or a catalyst induces a reaction to give rise to atransformation of the reagent to another reagent, intermediate, orproduct. A reagent may also undergo a reaction such as a phase change(e.g., precipitation) upon interaction with one or more components ofother plug-fluids or a reagent-containing carrier-fluid.

The term “substrate” refers to a layer or piece of material from whichdevices or chips are prepared or manufactured. As used herein, the term“substrate” includes any substrate fabricated using any traditional orknown microfabrication techniques. The term “substrate” also referseither to an entire device or chip or to a portion, area, or section ofa device or chip which may or may not be removable or detachable fromthe main body of the device or chip. The substrate may be prepared fromone or more materials such as glass, silicon, silicone elastomer, andpolymers including, but not limited to, polypropylene or polyethylene.

The discussion below provides a detailed description of various devicesand methods according to the invention for forming plugs, generatinggradients in a series of plugs, varying the concentration of reagentsinside plugs, rapid mixing in plugs, and scaling of mixing times. Inparticular, a detailed description of methods for merging, splittingand/or sorting plugs using channels, which form the bases for variousapplications ranging from the manufacture and analysis of variousproducts to applications in electronics, medicine, diagnostics, andpharmaceuticals, to name a few, is discussed. Methods of detection andmeasurement of, among others, plugs and processes occurring within plugsare also described.

Among the various applications involving the devices and methodsaccording to the invention are particle separation/sorting, synthesis,investigation of nonlinear and stochastic systems, nonlinearamplification using unstable autocatalytic mixtures, use of stochasticchemical systems for chemical amplification, kinetic measurements, timecontrol of processes, increasing the dynamic range of kineticmeasurements, ultrafast measurements, crystallization of proteins, anddynamic control of surface chemistry.

In addition, the devices and methods according to the invention offer awide-range of other applications. For example, the devices and methodsaccording to the invention provide for effective, rapid, and precisemanipulation and monitoring of solutions or reactions over a range oftime scales (e.g., from tens of microseconds, to hours or weeks in caseof, for example, crystallization) and over a range of solution volumes(e.g., from femtoliters to hundreds of nanoliters).

In one aspect of the invention, the various devices and methodsaccording to the invention are used to overcome one or more of thefollowing problems involving microfluidics. First, the substantialdispersion of solutes in microfluidic channels increases reagentconsumption and makes experiments or measurements over long time scales(e.g., minutes to hours) difficult to perform. Various devices andmethods according to the invention are intended to overcome this problemby localizing reagents inside plugs that are encapsulated by animmiscible carrier-fluid.

Second, slow mixing of solutions renders experiments, tests, orreactions involving very short time scales (e.g., tens of millisecondsand below) either difficult or impossible to perform with existingtechnologies. In addition, turbulence-based mixing techniquesprohibitively increase sample consumption. In accordance with thepresent invention, this problem is preferably addressed by conductingthe mixing process inside plugs. Rather than relying on turbulence, thevarious devices and methods according to the invention preferably relyon chaotic advection to accelerate the mixing process. An advantageprovided by chaotic advection is that it is expected to operateefficiently in both small and large channels.

Third, achieving control over the chemistry of internal surfaces ofdevices can be very important at small scales. Thus, being able tocontrol surface chemistry in small devices for example is highlydesirable. In accordance with the devices and methods according to theinvention, the surface chemistry to which solutions are exposed ispreferably controlled through a careful selection of surfactants thatare preferably designed to assemble at the interface between the plugsand the immiscible fluid that surrounds them.

Devices and methods of the invention are also provided for use intraditional areas of microfluidics where, for example, miniaturizationand speed are important. Thus, the devices and methods according to theinvention may be used to develop various tools such as those forhigh-throughput chemical or biophysical measurements, chemicalsynthesis, particle formation, and protein crystallization. They mayalso be used in high-throughput screening, combinatorial synthesis,analysis, and diagnostics, either as a self-contained platform, or incombination with existing technologies particularly those that rely onthe use of immiscible fluid flows.

Importantly, the devices of the invention can be adapted to work withautomation and robotic technology. They may be used, for example, as abasis for ultra-high throughput automated systems for structural andfunctional characterization of biological molecules. Thus, the variousdevices and methods according to the invention provide rapid,economical, and accessible means of synthesis, analysis, andmeasurements in the fields of biology, chemistry, biophysics,bioengineering, and medicine (e.g., for diagnostics).

The devices and methods of the invention have numerous other possibleapplications. For example, chaotic mixing at low values of Reynoldsnumber can be exploited as an important tool for controlling unstablechemical reactions. In addition, the systems and devices of theinvention may be used for controlling and/or monitoring reactions thatgenerate highly unstable (or explosive) intermediates. They can also bevaluable for controlling or monitoring reactions or processes involvingautocatalytic reactions. For example, pure hydrogen peroxide (H₂O₂) isan inexpensive and highly effective oxidant, but its autocatalyticdecomposition often leads to explosions upon storage and handling. Inthe microfluidic systems of the invention, H₂O₂ is preferably generatedin-situ, stabilized by the chaotic flow, and used to destroy chemicaland biological warfare agents. Because the unstable mixtures in thesesystems are localized inside plugs formed in accordance with theinvention, occasional autocatalytic decomposition in one or more plugsis kept localized within those plugs thereby preventing a catastrophicreaction involving the whole system. In addition, large arrays ofmicrofluidic reactors may be operated in parallel to provide substantialthroughput.

It is also possible to couple multiple autocatalytic reactions in asingle network using the devices and methods according to the invention.For example, a sample plug could be split into many smaller plugs andforwarded to individual amplification cascades. Because the contents ofthe cascades' outflows exhibit patterns that correspond to the patternsof analytes present in these systems, these patterns could be analyzedusing artificial neural network (ANN) (Jackson, R. B. a. T. NeuralComputing: An Introduction, Hilger, New York, 1991; Zornetzer et al., AnIntroduction to Neural and Electronic Networks, Academic Press, SanDiego, Calif., 1990.) algorithms. For example, patterns that arise inblood or saliva analysis may correspond to certain normal or abnormal(e.g., disease, fatigue, infection, poisoning) conditions involving, forexample, human and animals.

Moreover, it may be possible to create intelligent microfluidic systemsin accordance with the invention, where the nonlinear chemical reactionsperform not only detection, but also analysis using ANN algorithms. Forexample, after amplification, the channels of the present inventiontypically will contain sufficient amounts of material to operatehydrogel-based valves (Liu et al., “Fabrication and characterization ofhydrogel-based microvalves,” J. Microelectromech. Syst. 2002, vol. 1,pp. 45-53; Yu et al., “Responsive biomimetic hydrogel valve formicrofluidics,” Appl. Phys. Lett. 2001, vol. 78, pp. 2589-2591; Beebe etal., “Functional hydrogel structures for autonomous flow control insidemicrofluidic channels,” Nature, 2000, vol. 404, 588.). These valves canbe used to control flows inside the system as a function of the sampleplug composition. Feedforward and even feedback (e.g., by using thehydrogel valves to control the flow of the input streams) networks maythus be created and used for analysis. Such nonlinear networks may beused not only to recognize patterns pre-programmed by the connectivityof the channels (Hjelmfelt et al., “Pattern-Recognition in CoupledChemical Kinetic Systems,” Science, 1993, 260, 335-337.) but also tolearn patterns by reconfiguring themselves (Jackson, R. B. a. T. NeuralComputing: An Introduction, Hilger, New York, 1991; Zornetzer et al., AnIntroduction to Neural and Electronic Networks, Academic Press, SanDiego, Calif., 1990.). Such intelligent microfluidic devices could haveunprecedented capabilities for fully autonomous detection, analysis, andsignal processing, perhaps surpassing those of biological and currentman-made systems.

The devices and methods of the invention are also useful in genomics andproteomics, which are used to identify thousands of new biomoleculesthat need to be characterized, or are available only in minutequantities. In particular, the success of genomics and proteomics hasincreased the demand for efficient, high-throughput mechanisms forprotein crystallization. X-ray structure determination remains thepredominant method of structural characterization of proteins. However,despite significant efforts to understand the process ofcrystallization, macromolecular crystallization largely remains anempirical field, with no general theory to guide a rational approach. Asa result, empirical screening has remained the most widely used methodfor crystallizing proteins.

The following areas also provide applications of the devices and methodsaccording to the invention. For example, a number of problems stillbeset high-throughput kinetics and protein crystallization. When itcomes to determining protein structure and quantitatively ascertainingprotein interactions, there are at least two technological challenges:(1) most robotic technology still only automate existing methods and areoften too expensive for a small research laboratory; and (2) thereremains the need for conceptually new methods that provide greaterdegree of control over the crystallization process. In addition, settingup and monitoring crystallization trials typically involve handling ofsub-microliter volumes of fluids over periods ranging from seconds todays.

Thus, various devices and methods according to the present invention aredesigned to provide novel and efficient means for high-throughputcrystallization of soluble and membrane proteins. In addition to being asimple and economical method of setting up thousands of crystallizationtrials in a matter of minutes, a system according to the invention willenable unique time control of processes such as the mixing andnucleation steps leading to crystallization. A system according to thepresent invention may also be used to control protein crystallization bycontrolling not only short time-scale events such as nucleation but alsolong time-scale events such as crystal growth.

Further, the devices and methods of the present invention may be used inhigh-throughput, kinetic, and biophysical measurements spanning the10⁻⁵-10⁷ second time regime. Preferably, the various devices and methodsaccording to the present invention require only between about a fewnanoliters to about a few microliters of each solution. Applications ofsuch devices and methods include studies of enzyme kinetics and RNAfolding, and nanoparticle characterization and synthesis, which arediscussed in detail below.

Channels and Devices

In one aspect of the invention, a device is provided that includes oneor more substrates comprising a first channel comprising an inletseparated from an outlet; optionally, one or more secondary channels (orbranch channels) in fluid communication with the first channel, at leastone carrier-fluid reservoir in fluid communication with the firstchannel, at least two plug-fluid reservoirs in fluid communication withthe first channel, and a means for applying continuous pressure to afluid within the substrate.

A device according to the invention preferably comprises at least onesubstrate.

A substrate may include one or more expansions or areas along a channelwherein plugs can be trapped. The substrates of the present inventionmay comprise an array of connected channels.

The device may have one or more outlet ports or inlet ports. Each of theoutlet and inlet ports may also communicate with a well or reservoir.The inlet and outlet ports may be in fluid communication with thechannels or reservoirs that they are connecting or may contain one ormore valves. Fluid can be introduced into the channels via the inlet byany means. Typically, a syringe pump is used, wherein the flow rate ofthe fluid into the inlet can be controlled.

A plug-forming region generally comprises a junction between aplug-fluid inlet and a channel containing the carrier-fluid such thatplugs form which are substantially similar in size to each other andwhich have cross-sections which are substantially similar in size to thecross-section of the channel in the plug-forming region. In oneembodiment, the substrate may contain a plurality of plug-formingregions.

The different plug-forming regions may each be connected to the same ordifferent channels of the substrate. Preferably, the sample inletintersects a first channel such that the pressurized plug fluid isintroduced into the first channel at an angle to a stream ofcarrier-fluid passing through the first channel. For example, inpreferred embodiments, the sample inlet and first channel intercept at aT-shaped junction; i.e., such that the sample inlet is perpendicular(i.e. at an angle of 90°) to the first channel. However, the sampleinlet may intercept the first channel at any angle.

A first channel may in turn communicate with two or more branch channelsat another junction or “branch point”, forming, for example, a T-shapeor a Y-shape. Other shapes and channel geometries may be used asdesired. In exemplary embodiments the angle between intersectingchannels is in the range of from about 60° to about 120°. Particularexemplary angles are 45°, 60°, 90°, and 120°. Precise boundaries for thediscrimination region are not required, but are preferred.

The first and branch channels of the present invention can, eachindependently, be straight or have one or more bends. The angle of abend, relative to the substrate, can be greater than about 10°,preferably greater than about 135°, 180°, 270°, or 360°.

In one embodiment of the invention, a substrate comprises at least oneinlet port in communication with a first channel at or near aplug-forming region, a detection region within or coincident with all ora portion of the first channel or plug-forming region, and a detectorassociated with the detection region. In certain embodiments the devicemay have two or more plug-forming regions. For example, embodiments areprovided in which the analysis unit has a first inlet port incommunication with the first channel at a first plug-forming region, asecond inlet port in communication with the first channel at a secondplug-forming region (preferably downstream from the first plug-formingregion), and so forth.

In another embodiment, a substrate according to the invention maycomprise a first channel through which a pressurized stream or flow of acarrier-fluid is passed, and two or more inlet channels which intersectthe first channel at plug-forming regions and through which apressurized stream or flow of plug fluids pass. Preferably, these inletchannels are parallel to each other and each intercept the first channelat a right angle. In specific embodiments wherein the plugs introducedthrough the different plug forming regions are mixed, the inlet channelsare preferably close together along the first channel. For example, thefirst channel may have a diameter of 60 μm that tapers to 30 μm at ornear the plug-forming regions. The inlet channels then also preferablyhave a diameter of about 30 μm and, in embodiments where plug mixing ispreferred, are separated by a distance along the first channelapproximately equal to the diameter of the inlet channel (i.e., about 30μm).

In an embodiment according to the invention, the substrate also has adetection region along a channel. There may be a plurality of detectionregions and detectors, working independently or together, e.g., toanalyze one or more properties of a chemical such as a reagent.

A detection region is within, communicating, or coincident with aportion of a first channel at or downstream of the plug-forming regionand, in sorting embodiments, at or upstream of the discrimination regionor branch point. Precise boundaries for the detection region are notrequired, but are preferred.

A typical substrate according to the invention comprises a carrier-fluidinlet that is part of and feeds or communicates directly with a firstchannel, along with one or more plug fluid inlets in communication withthe first channel at a plug-forming region situated downstream from themain inlet (each different plug-fluid inlet preferably communicates withthe first channel at a different plug-forming region).

Plugs formed from different plug-fluids or solutions may be released inany order. For example, an aqueous solution containing a firstplug-fluid may be released through a first inlet at a first plug-formingregion. Subsequently, plugs of an aqueous second plug-fluid may bereleased through a second inlet at a second plug-forming regiondownstream of the first inlet.

Fabrication of Channels, Substrates, and Devices

The substrates and devices according to the invention are fabricated,for example by etching a silicon substrate, chip, or device usingconventional photolithography techniques or micromachining technology,including soft lithography. The fabrication of microfluidic devicesusing polydimethylsiloxane has been previously described. These andother fabrication methods may be used to provide inexpensiveminiaturized devices, and in the case of soft lithography, can providerobust devices having beneficial properties such as improvedflexibility, stability, and mechanical strength. Preferably, whenoptical detection is employed, the invention also provides minimal lightscatter from, for example, plugs, carrier-fluid, and substrate material.Devices according to the invention are relatively inexpensive and easyto set up.

Machining methods (e.g., micromachining methods) that may be used tofabricate channels, substrates, and devices according to the inventionare well known in the art and include film deposition processes, such asspin coating and chemical vapor deposition, laser fabrication orphotolithographic techniques, or etching methods, which may be performedeither by wet chemical or plasma processes.

Channels may be molded onto optically transparent silicone rubber orpolydimethylsiloxane (PDMS), preferably PDMS. This can be done, forexample, by casting the channels from a mold by etching the negativeimage of these channels into the same type of crystalline silicon waferused in semiconductor fabrication. The same or similar techniques forpatterning semiconductor features can be used to form the pattern of thechannels. In one method of channel fabrication, an uncured PDMS ispoured onto the molds placed in the bottom of, for example, a Petridish. To accelerate curing, the molds are preferably baked. After curingthe PDMS, it is removed from on top of the mold and trimmed. Holes maybe cut into the PDMS using, for example, a tool such as a cork borer ora syringe needle. Before use, the PDMS channels may be placed in a hotbath of HCl if it is desired to render the surface hydrophilic. The PDMSchannels can then be placed onto a microscope cover slip (or any othersuitable flat surface), which can be used to form the base/floor or topof the channels.

A substrate according to the invention is preferably fabricated frommaterials such as glass, polymers, silicon microchip, or siliconeelastomers. The dimensions of the substrate may range, for example,between about 0.3 cm to about 7 cm per side and about 1 micron to about1 cm in thickness, but other dimensions may be used.

A substrate can be fabricated with a fluid reservoir or well at theinlet port, which is typically in fluid communication with an inletchannel. A reservoir preferably facilitates introduction of fluids intothe substrate and into the first channel. An inlet port may have anopening such as in the floor of the substrate to permit entry of thesample into the device. The inlet port may also contain a connectoradapted to receive a suitable piece of tubing, such as Teflon® tubing,liquid chromatography or HPLC tubing, through which a fluid may besupplied. Such an arrangement facilitates introducing the fluid underpositive pressure in order to achieve a desired pressure at theplug-forming region.

A substrate containing the fabricated flow channels and other componentsis preferably covered and sealed, preferably with a transparent cover,e.g., thin glass or quartz, although other clear or opaque covermaterials may be used. Silicon is a preferred substrate material due towell-developed technology permitting its precise and efficientfabrication, but other materials may be used, including polymers such aspolytetrafluoroethylenes. Analytical devices having channels, valves,and other elements can be designed and fabricated from various substratematerials. When external radiation sources or detectors are employed,the detection region is preferably covered with a clear cover materialto allow optical access to the fluid flow. For example, anodic bondingof a silicon substrate to a PYREX® cover slip can be accomplished bywashing both components in an aqueous H₂SO₄/H₂O₂ bath, rinsing in water,and then, for example, heating to about 350° C. while applying a voltageof 450 V.

A variety of channels for sample flow and mixing can be fabricated onthe substrate and can be positioned at any location on the substrate,chip, or device as the detection and discrimination or sorting points.Channels can also be designed into the substrate that place the fluidflow at different times/distances into a field of view of a detector.Channels can also be designed to merge or split fluid flows at precisetimes/distances.

A group of manifolds (a region consisting of several channels that leadto or from a common channel) can be included to facilitate the movementof plugs from different analysis units, through the plurality of branchchannels and to the appropriate solution outlet. Manifolds arepreferably fabricated into the substrate at different depth levels.Thus, devices according to the invention may have a plurality ofanalysis units that can collect the solution from associated branchchannels of each unit into a manifold, which routes the flow of solutionto an outlet. The outlet can be adapted for receiving, for example, asegment of tubing or a sample tube, such as a standard 1.5 ml centrifugetube. Collection can also be done using micropipettes.

Methods of Forming Plugs

The various channels, substrates, and devices according to the inventionare primarily used to form and manipulate plugs.

In a preferred embodiment, plug-fluids do not significantly mix at orbefore they are introduced into the first channel. The plug-fluids mayform distinct laminar streams at or before the inlet. They may beseparated by an additional fluid. Alternatively, they may be introducedinto the carrier-fluid via inlets of differing size. The concentrationof plug-fluids in the plugs may be adjusted by adjusting volumetric flowrates of the plug-fluids. Further, the diameters of the first channeland the branch channel(s) may differ.

FIGS. 2A(1) and 2A(2) are schematic diagrams contrasting laminar flowtransport and plug transport in a channel, respectively. In FIG. 2A(2)(the lower figure which depicts the transport of plugs), two aqueousreagents (marked in red and blue) form laminar streams that areseparated by a “divider” aqueous stream. The three streams enter achannel with flowing oil, at which point plugs form and plug fluids mix.During plug transport, rapid mixing of the plug-fluids typically occurswithin the plugs. In contrast, in laminar flow transport, fluid mixingoccurs slowly, and with high dispersion, as shown in FIG. 2A(1) (theupper figure). In FIG. 2A(1) (the upper figure), the time t at a givenpoint d₁ can be estimated from t₁≈d₁/U, where dl is the distance fromd=0 and U is the flow velocity. In FIG. 2A(2) (the lower figure), thetime t is given by t₁=d₁/U.

FIG. 2B shows a photograph and a schematic diagram that depict mixing inwater/oil plugs (upper schematic and photograph) and in laminar streams(lower schematic and photograph) comprising only aqueous plug-fluids.The oil (carrier-fluid in this case) is introduced into channel 200 of asubstrate. Instead of oil, water is introduced into the correspondingchannel 207 in the case of mixing using laminar streams. The threeaqueous plug-fluids are introduced by inlet ports 201, 202, 203 into thecarrier-fluid (and by inlet ports 204, 205, 206 in the case of laminarstreams). A preferred scheme is one in which the aqueous plug-fluidsinitially coflow preferably along a short or minimal distance beforecoming in contact with the carrier-fluid. In a preferred embodiment, thedistance traversed by the coflowing plug-fluids is approximately orsubstantially equal to the width of the channel.

The middle or second aqueous plug-fluid in the top figure may be plainwater, buffer, solvent, or a different plug-fluid. The middle aqueousplug-fluid would preferably initially separate the two other aqueousplug-fluids before the aqueous fluids come into contact with thecarrier-fluid. Thus, the intervening aqueous plug-fluid would prevent,delay, or minimize the reaction or mixing of the two outer aqueousplug-fluids before they come in contact with the carrier-fluid. Theplugs that form in the plug-forming region can continue along anunbranched channel, can split and enter a channel, can merge with plugsfrom another channel, or can exit the substrate through an exit port. Itcan be seen in FIGS. 2A(1) and 2A(2) that, in the absence of an oil, theaqueous plug-fluids flow in laminar streams without significant mixingor with only partial mixing. In contrast, plug-fluids mix substantiallyor completely in the plugs.

FIGS. 3A and 3B show photographs and schematic diagrams that depict astream of plugs from an aqueous plug-fluid and an oil (carrier-fluid) incurved channels at flow rates of 0.5 μL/min (FIG. 3A top schematicdiagram and photograph) and 1.0 μL/min (FIG. 3B bottom schematic diagramand photograph). This scheme allows enhanced mixing of reagents in theelongated plugs flowing along a curved channel with smooth corners orcurves. The carrier-fluid is introduced into an inlet port 300, 307 of asubstrate while the three aqueous plug-fluids are introduced in separateinlet ports 301-306. As in FIGS. 2(A)(1)-2(A)(2) and 2(B)(1)-2(B)(2), apreferred scheme would be one in which the plug-fluids initially coflowpreferably along a short or minimal distance before coming in contactwith the carrier-fluid. In a preferred embodiment, the distancetraversed by the coflowing plug-fluids (e.g., aqueous plug-fluids) isapproximately or substantially equal to the width of the channel. Themiddle or second aqueous plug-fluid may comprise plain water, buffer,solvent, or a plug-fluid, and the middle aqueous plug-fluid preferablyinitially separates the two other aqueous plug-fluids before the aqueousplug-fluids come into contact with the carrier-fluid which, in thiscase, is an oil. Thus, the intervening aqueous plug-fluid would prevent,delay, or minimize the reaction or mixing of the two outer aqueousplug-fluids before they come in contact with the oil (or carrier-fluid).

FIG. 4 shows a photograph and schematic diagram that illustrate plugformation through the injection of oil and multiple plug-fluids.Although FIG. 4 shows five separate plug-fluids, one may also separatelyintroduce less than or more than five plug-fluids into the substrate.The reagents or solvents comprising the plug-fluids may be different orsome of them may be identical or similar. As in FIG. 2, the oil isintroduced into an inlet port 400 of a substrate while the aqueousplug-fluid is introduced in separate inlet ports 401-405. The waterplugs then flow through exit 406. A preferred scheme is one in which theaqueous plug-fluids would initially coflow preferably along a short orminimal distance before coming in contact with the oil. In a preferredembodiment, the distance traversed by the coflowing plug-fluids isapproximately or substantially equal to the width of the channel. One ormore of the aqueous plug-fluids may comprise plain water, buffer,solvent, or a plug-fluid, and at least one aqueous plug-fluid wouldpreferably initially separate at least two other aqueous streams beforethe aqueous plug-fluid comes into contact with the oil. Thus, the atleast one intervening aqueous plug-fluid would prevent, delay, orminimize the reaction or mixing of the two outer aqueous streams beforethe aqueous streams come in contact with the oil. FIG. 5 shows amicrofluidic network, which is similar to that shown in FIG. 4, in whichseveral reagents can be introduced into the multiple inlets. Inaddition, FIG. 5 shows a channel having a winding portion through whichthe plugs undergo mixing of the four reagents A, B, C, and D. As shownin FIG. 5, the reagents A, B, C, and D are introduced into inlet ports501, 503, 505, and 507, while aqueous streams are introduced into inletports 502, 504, 506. FIG. 5 shows plugs through the various stages ofmixing, wherein mixture 50 corresponds to the initial A+B mixture,mixture 51 corresponds to the initial C+D mixture, mixture 52corresponds to the mixed A+B mixture, mixture 53 corresponds to themixed C+D mixture, and mixture 54 corresponds to the A+B+C+D mixture.

The formation of the plugs preferentially occurs at low values of thecapillary number C.n., which is given by the equation

C.n.=Uμ/γ  Eqn. (1)

where U is the flow velocity, It is the viscosity of the plug fluid orcarrier-fluid, and γ is the surface tension at the water/surfactantinterface.

The plugs may be formed using solvents of differing or substantiallyidentical viscosities. Preferably, the conditions and parameters used inan experiment or reaction are such that the resulting capillary numberlies in the range of about 0.001≤C.n.≤about 10. Preferably, the valuesof parameters such as viscosities and velocities are such that plugs canbe formed reliably. Without wishing to be bound by theory, it isbelieved that as long as flow is not stopped, the C.n. is ≤about 0.2,and as long as the surface tension of the plug-fluid/carrier-fluidinterface is lower than the surface tension of the solution/wallinterface, plug formation will persist. The C.n. number is zero whenflow is stopped.

In one embodiment, in which perfluorodecaline was used as thecarrier-fluid and the plug-fluid was aqueous, it was found that thissystem can be operated at values of C.n. up to ˜0.1 (at 300 mm s⁻¹). Inthis system, as the value of the C.n. increased above 0.2, the formationof plugs became irregular. The viscosity of perfluorodecaline is5.10×10⁻³ kg m⁻³ s⁻¹, the surface tension at the interface between theplugs and the carrier-fluid was 13×10³ N m⁻¹.

The length of the plugs can be controlled such that their sizes canrange from, for example, about 1 to 4 times a cross-sectional dimension(d, where d is a channel cross-sectional dimension) of a channel usingtechniques such as varying the ratio of the plug-fluids andcarrier-fluids or varying the relative volumetric flow rates of theplug-fluid and carrier-fluid streams. Short plugs tend to form when theflow rate of the aqueous stream is lower than that of a carrier-fluidstream. Long plugs tend to form when the flow rate of the plug-fluidstream is higher than that of the carrier stream.

In one approximation, the volume of a plug is taken equal to about 2×d³,where d is a cross-sectional dimension of a channel. Thus, the plugs canbe formed in channels having cross-sectional areas of, for example, from20×20 to 200×200 μm², which correspond to plug volumes of between about16 picoliters (pL) to 16 nanoliters (nL). The size of channels may beincreased to about 500 μm (corresponding to a volume of about 250 nL) ormore. The channel size can be reduced to, for example, about 1 μm(corresponding to a volume of about 1 femtoliter). Larger plugs areparticularly useful for certain applications such as proteincrystallizations, while the smaller plugs are particularly useful inapplications such as ultrafast kinetic measurements.

In one preferred embodiment, plugs conform to the size and shape of thechannels while maintaining their respective volumes. Thus, as plugs movefrom a wider channel to a narrower channel they preferably become longerand thinner, and vice versa.

Plug-fluids may comprise a solvent and optionally, a reactant. Suitablesolvents for use in the invention, such as those used in plug-fluids,include organic solvents, aqueous solvents, oils, or mixtures of thesame or different types of solvents, e.g. methanol and ethanol, ormethanol and water. The solvents according to the invention includepolar and non-polar solvents, including those of intermediate polarityrelative to polar and non-polar solvents. In a preferred embodiment, thesolvent may be an aqueous buffer solution, such as ultrapure water(e.g., 18 MΩ resistivity, obtained, for example, by columnchromatography), 10 mM Tris HCl, and 1 mM EDTA (TE) buffer, phosphatebuffer saline or acetate buffer. Other solvents that are compatible withthe reagents may also be used.

Suitable reactants for use in the invention include synthetic smallmolecules, biological molecules (i.e., proteins, DNA, RNA,carbohydrates, sugars, etc.), metals and metal ions, and the like.

The concentration of reagents in a plug can be varied. In one embodimentaccording to the invention, the reagent concentration may be adjusted tobe dilute enough that most of the plugs contain no more than a singlemolecule or particle, with only a small statistical chance that a plugwill contain two or more molecules or particles. In other embodiments,the reagent concentration in the plug-fluid is adjusted to concentrateenough that the amount of reaction product can be maximized.

Suitable carrier-fluids include oils, preferably fluorinated oils.Examples include viscous fluids, such as perfluorodecaline orperfluoroperhydrophenanthrene; nonviscous fluids such asperfluorohexane; and mixtures thereof (which are particularly useful formatching viscosities of the carrier-fluids and plug-fluids).Commercially available fluorinated compounds such as Fluorinert™ liquids(3M, St. Paul, Minn.) can also be used.

The carrier-fluid or plug-fluid, or both may contain additives, such asagents that reduce surface tensions (e.g., surfactants). Other agentsthat are soluble in a carrier-fluid relative to a plug-fluid can also beused when the presence of a surfactant in the plug fluid is notdesirable. Surfactants may be used to facilitate the control andoptimization of plug size, flow and uniformity. For example, surfactantscan be used to reduce the shear force needed to extrude or inject plugsinto an intersecting channel. Surfactants may affect plug volume orperiodicity, or the rate or frequency at which plugs break off into anintersecting channel. In addition, surfactants can be used to controlthe wetting of the channel walls by fluids. In one embodiment accordingto the invention, at least one of the plug-fluids comprises at least onesurfactant.

Preferred surfactants that may be used include, but are not limited to,surfactants such as those that are compatible with the carrier andplug-fluids. Exemplary surfactants include Tween™, Span™, andfluorinated surfactants (such as Zonyl™ (Dupont, Wilmington Del.)). Forexample, fluorinated surfactants, such as those with a hydrophilic headgroup, are preferred when the carrier-fluid is a fluorinated fluid andthe plug-fluid is an aqueous solution.

However, some surfactants may be less preferable in certainapplications. For instance, in those cases where aqueous plugs are usedas microreactors for chemical reactions (including biochemicalreactions) or are used to analyze and/or sort biomaterials, a watersoluble surfactant such as SDS may denature or inactivate the contentsof the plug.

The carrier-fluid preferably wets the walls of the channelspreferentially over the plugs. If this condition is satisfied, the plugtypically does not come in contact with the walls of the channels, andinstead remains separated from the walls by a thin layer of thecarrier-fluid. Under this condition, the plugs remain stable and do notleave behind any residue as they are transported through the channels.The carrier-fluid's preferential wetting of the channel walls over theplug-fluid is achieved preferably by setting the surface tension by, forexample, a suitable choice of surfactant. Preferably, the surfacetension at a plug fluid/channel wall interface (e.g., about 38 mN/msurface tension for a water/PDMS interface) is set higher than thesurface tension at a plug fluid/carrier-fluid interface (e.g., about 13mN/m for a water/carrier-fluid interface with a surfactant such as 10%1H,1H,2H,2H-perfluorooctanol in perfluorodecaline as the carrier-fluid).If this condition is not satisfied, plugs tend to adhere to the channelwalls and do not undergo smooth transport (e.g., in the absence of1H,1H,2H,2H-perfluorooctanol the surface tension at thewater/perfluorodecaline interface is about 55 mN/m, which is higher thanthe surface tension of the water/PDMS interface (e.g., about 38 mN/m)),and plugs adhere to the walls of the PDMS channels. Because the walls ofthe channels (PDMS, not fluorinated) and the carrier-fluid (fluorinatedoil) are substantially different chemically, when a fluorinatedsurfactant is introduced, the surfactant reduces the surface tension atthe oil-water interface preferentially over the wall-water interface.This allows the formation of plugs that do not stick to the channelwalls.

The surface tension at an interface may be measured using what is knownas a hanging drop method, although one may also use other methods.Preferably, the surface tension is sufficiently high to avoiddestruction of the plugs by shear.

The plug-fluids and carrier-fluids may be introduced through one or moreinlets. Specifically, fluids may be introduced into the substratethrough pneumatically driven syringe reservoirs that contain either theplug-fluid or carrier-fluid. Plugs may be produced in the carrier-fluidstream by modifying the relative pressures such that the plug-fluidscontact the carrier-fluid in the plug-forming regions then shear offinto discrete plugs.

In the invention, plugs are formed by introducing the plug-fluid, at theplug-forming region, into the flow of carrier-fluid passing through thefirst channel. The force and direction of flow can be controlled by anydesired method for controlling flow, for example, by a pressuredifferential, or by valve action. This permits the movement of the plugsinto one or more desired branch channels or outlet ports.

In preferred embodiments according to the invention, one or more plugsare detected, analyzed, characterized, or sorted dynamically in a flowstream of microscopic dimensions based on the detection or measurementof a physical or chemical characteristic, marker, property, or tag.

The flow stream in the first channel is typically, but not necessarilycontinuous and may be stopped and started, reversed or changed in speed.Prior to sorting, a non-plug-fluid can be introduced into a sample inletport (such as an inlet well or channel) and directed through theplug-forming region, e.g., by capillary action, to hydrate and preparethe device for use. Likewise, buffer or oil can also be introduced intoa main inlet port that communicates directly with the first channel topurge the substrate (e.g., of “dead” air) and prepare it for use. Ifdesired, the pressure can be adjusted or equalized, for example, byadding buffer or oil to an outlet port.

The pressure at the plug-forming region can also be regulated byadjusting the pressure on the main and sample inlets, for example withpressurized syringes feeding into those inlets. By controlling thedifference between the oil and water flow rates at the plug-formingregion, the size and periodicity of the plugs generated may beregulated. Alternatively, a valve may be placed at or coincident toeither the plug-forming region or the sample inlet connected thereto tocontrol the flow of solution into the plug-forming region, therebycontrolling the size and periodicity of the plugs. Periodicity and plugvolume may also depend on channel diameter and/or the viscosity of thefluids.

Mixing in Plugs

FIGS. 7 (a)-(b) show microphotographs (10 μs exposure) illustratingrapid mixing inside plugs (a) and negligible mixing in a laminar flow(b) moving through winding channels at the same total flow velocity.Aqueous streams were introduced into inlets 700-705 in FIGS. 7(a)-(b).In FIGS. 7(c) and 7(e), Fluo-4 was introduced into inlets 706, 709,buffer was introduced into inlets 707, 710, and CaCl₂ was introducedinto inlets 708, 711. FIG. 7(c) shows a false-color microphotograph (2 sexposure, individual plugs are invisible) showing time-averagedfluorescence arising from rapid mixing inside plugs of solutions ofFluo-4 (54 μM) and CaCl₂ (70 μM) in aqueous sodium morpholinepropanesulfonate buffer (20 μM, pH 7.2); this buffer was also used asthe middle aqueous stream. FIGS. 7(d)(i) and 7(d)(ii) show plots of therelative normalized intensity (I) of fluorescence obtained from imagessuch as shown in (c) as a function of distance (left, FIG. 7(d)(i))traveled by the plugs and of time required to travel that distance(right, FIG. 7(d)(ii)) at a given flow rate. The total intensity acrossthe width of the channel was measured. Total PFD/water volumetric flowrates (in μL min⁻¹) were 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6, and 20:6.FIG. 7(e) shows a false-color microphotograph (2 s exposure) of the weakfluorescence arising from negligible mixing in a laminar flow of thesolutions used in (c). All channels were 45 μm deep; inlet channels were50 μm and winding channels 28 μm wide; Re˜5.3 (water), ˜2.0 (PFD).

FIGS. 8(a)-8(d) show photographs and schematics that illustrate fastmixing at flow rates of about 0.5 μL/min (top schematic diagram andphotograph, FIGS. 8(a) and (b)) and about 1.0 μL/min (lower schematicdiagram and photograph, FIGS. 8(c) and (d)) using 90°-step channelswhile FIGS. 9(a)-9(d) illustrate fast mixing at flow rates of about 1.0μL/min (top schematic diagram and photograph, FIGS. 9(a) and (b)) andabout 0.5 μL/min (lower schematic diagram and photograph, FIGS. 9(c) and(d)) using 135°-step channels. Aqueous streams are introduced intoinlets 800-805 in FIGS. 8(a) and (c) (inlets 900-905 in FIGS. 9(a) and(c)), while a carrier fluid is introduced into channels 806, 807(channels 906, 907 in FIGS. 9(a) and (c)). The plugs that form then flowthrough exits 808, 809 (FIGS. 8(a) and (c)) and exits 908, 909 (FIGS.9(a) and (c)). As can be seen in FIGS. 8(b) and (d) and FIGS. 9(b) and(d), the plugs are transported along multi-step channels, instead ofchannels with smooth curves (as opposed to channels with sharp corners).An advantage of these multi-step configurations of channels is that theymay provide further enhanced mixing of the substances within the plugs.

Several approaches may be used to accelerate or improve mixing. Theseapproaches may then be used to design channel geometries that allowcontrol of mixing. Flow can be controlled by perturbing the flow insidea moving plug so that it differs from the symmetric flow inside a plugthat moves through a straight channel. For example, flow perturbationcan be accomplished by varying the geometry of a channel (e.g., by usingwinding channels), varying the composition of the plug fluid (e.g.,varying the viscosities), varying the composition of the carrier-fluid(e.g., using several laminar streams of carrier-fluids that aredifferent in viscosity or surface tension to form plugs; in this case,mixing is typically affected, and in some cases enhanced), and varyingthe patterns on the channel walls (e.g., hydrophilic and hydrophobic, ordifferentially charged, patches would interact with moving plugs andinduce time-periodic flow inside them, which should enhance mixing).

Various channel designs can be implemented to enhance mixing in plugs.FIG. 1A shows a schematic of a basic channel design, while FIG. 1B showsa series of periodic variations of the basic channel design. FIG. 1Cshows a series of aperiodic combinations resulting from a sequence ofalternating elements taken from a basic design element shown in FIG. 1Aand an element from the periodic variation series shown in FIGS.1B(1)-(4). When the effects of these periodic variations are visualized,aperiodic combinations of these periodic variations are preferably usedto break the symmetries arising from periodic flows (see FIG. 1C). Here,the relevant parameters are channel width, period, radius of curvature,and sequence of turns based on the direction of the turns. Theparameters of the basic design are defined such that c is the channelwidth, 1 is the period, and r is the radius of curvature. For the basicdesign, the sequence can be defined as (left, right, left, right), whereleft and right is relative to a centerline along the path taken by aplug in the channel.

FIGS. 1B(1)-4) show schematic diagrams of a series of periodicvariations of the basic design. At least one variable parameter ispreferably defined based on the parameters defined in FIG. 1a ). In FIG.1B(1), the channel width is c/2; in FIG. 1B(2), the period is 2 l; andin FIG. 1B(3), and the radius of curvature is 2r. In FIG. 1B(4), theradius of curvature is r/2 and the sequence is (left, left, right,right).

FIGS. 1C(1)-(4) show a schematic diagram of a series of aperiodiccombinations formed by combining the basic design element shown in FIG.1A with an element from the series of periodic variations in FIG.1B(1)-(4). In FIG. 1C(1), the alternating pattern of a period of thebasic design shown in FIG. 1A (here denoted as “a”) and a period of thechannel in FIG. 1B(1) (here denoted as “b1”) is given by a+b1+a+ . . .In FIG. 1C(2), the aperiodic combination is given by a+b2+a. In thechannel shown in FIG. 1C(3) (here denoted as “c3”), the aperiodiccombination is given by a+c3+a. In the channel shown in FIG. 1C(4) (heredenoted as “c4”), a (right, left) sequence is introduced with a kink inthe pattern. A repeating (left, right) sequence would normally beobserved. By adding this kink, the sequence becomes (left, right, left,right)+(right, left)+(left, right, left, right).

Another approach for accelerating mixing relies on rationally-designedchaotic flows on a microfluidic chip using what is known as the baker'stransformation. Reorientation of the fluid is critical for achievingrapid mixing using the baker's transformation. The baker'stransformation leads to an exponential decrease of the striationthickness (the distance over which mixing would have to occur bydiffusion) of the two components via a sequence of stretching andfolding operations. Typically, every stretch-fold pair reduces thestriation thickness by a factor of 2, although this factor may have adifferent value. The striation thickness (ST) can be represented, in anideal case, by Eqn. (2) below. Thus, in the ideal case, in a sequence ofn stretch-fold-reorient operations, the striation thickness undergoes anexponential decrease given by

ST(t _(n))=ST(t ₀)×2^(−n)  Eqn. (2)

where ST(t_(n)) represents the striation thickness at time t_(n), ST(t₀)represents the initial striation thickness at time t₀, and n is thenumber of stretch-fold-reorient operations.

In accordance with the invention, the baker's transformation ispreferably implemented by creating channels composed of a sequence ofstraight regions and sharp turns. FIG. 11 shows a schematic diagram of achannel geometry designed to implement and visualize the baker'stransformation of plugs flowing through microfluidic channels. Otherdesigns could also be used. The angles at the channel bends and thelengths of the straight portions are chosen so as to obtain optimalmixing corresponding to the flow patterns shown. Different lengths ofstraight paths and different turns may be used depending on theparticular application or reaction involved.

A plug traveling through every pair of straight part 112 and sharp-turnpart 111 of the channel, which is equivalent to one period of a baker'stransformation, will experience a series of reorientation, stretchingand folding. In a straight part of the channel, a plug will experiencethe usual recirculating flow. At a sharp turn, a plug normally rolls andreorients due to the much higher pressure gradient across the sharpinternal corner and also due to larger travel path along the outsidewall. This method of mixing based on the baker's transformation is veryefficient and is thus one of the preferred types of mixing. Inparticular, this type of mixing leads to a rapid reduction of the timerequired for reagent mixing via diffusion.

It is believed that plug formation can be maintained at about the sameflow rate in channels of different sizes because the limit of a flowrate is typically set by the capillary number, C.n., which isindependent of the channel size. At a fixed flow rate, the mixing timet_(mix) may decrease as the size of the channel (d) is reduced. First,it is assumed that it takes the same number n of stretch-fold-reorientcycles to mix reagents in both large and small channels. This assumption(e.g., for n˜5) is in approximate agreement with previously measuredmixing in d=55 and d=20 micrometer (μm) channels. Each cycle requires aplug to travel over a distance of approximately 2 lengths of the plug(approximately 3d). Therefore, mixing time is expected to beapproximately equal to the time it takes to travel 15d, and willdecrease linearly with the size of the channel, t_(mix)˜d. A method thatprovides mixing in about 1 ms in 25-μm channels preferably providesmixing in about 40 μs in 1-μm channels. Achieving microsecond mixingtimes generally requires the use of small channels. High pressures arenormally required to drive a flow through small channels.

Without wishing to be bound by theory, theoretical modeling indicatesthat the number of cycles it takes for mixing to occur in a channel withdiameter d is given approximately by

n×2^(2n) ≈dU/D  Eqn. (3)

where n is the number of cycles, U is the flow velocity, D is thediffusion constant, one cycle is assumed to be equal to 6d, and mixingoccurs when convection and diffusion time scales are matched. The mixingtime is primarily determined by the number of cycles. This resultindicates that mixing will be accelerated more than just in directproportion to the channel diameter. For example, when d decreases by afactor of 10, mixing time decreases by a factor of d×Log(d)=10×Log(10).With properly designed channels, mixing times in 1-μm channels can belimited to about 20 μs. Even at low flow rates or long channels (such asthose involving protein crystallization), however, significant mixingcan still occur. In addition, without being bound by theory, it isexpected that increasing the flow rate U by a factor of 10 will decreasethe mixing time by a factor of Log(U)/U=(Log (10))/10.

To visualize mixing in a channel according to the invention, a coloredmarker can be used in a single plug-fluid. The initial distribution ofthe marker in the plug has been observed to depend strongly on thedetails of plug formation. As the stationary aqueous plug was extrudedinto the flowing carrier-fluid, shearing interactions between the flowof the carrier-fluid and the plug-fluid induced an eddy thatredistributed the solution of the marker to different regions of theplug. The formation of this eddy is referred to here as “twirling” (seeFIG. 27b )). Twirling is not a high Reynolds number (Re) phenomenon (seeFIG. 30) since it was observed at substantially all values of Re and atsubstantially all velocities. However, the flow pattern of this eddyappears to be slightly affected by the velocity.

Various characteristics and behavior of twirling were observed. Twirlingredistributed the marker by transferring it from one side of the plug tothe other, e.g., from the right to the left side of the plug. The mostefficient mixing was observed when there was minimal fluctuations inintensity, i.e., when the marker was evenly distributed across the plug.While twirling was present during the formation of plugs of all lengthsthat were investigated, its significance to the mixing process appearsto depend on the length of the plug. For example, the extent of twirlingwas observed to be significantly greater for short plugs than for longplugs. Twirling was also observed to affect only a small fraction of thelong plugs and had a small effect on the distribution of the marker inthe plugs. Moreover, twirling occurred only at the tip of the formingplug before the tip made contact with the right wall of themicrochannel. Also, the amount of twirling in a plug was observed to berelated to the amount of the carrier-fluid that flowed past the tip. Theresults of experiments involving twirling and its effect on mixing showthat twirling is one of the most important factors, if not the mostimportant factor, in determining the ideal conditions for mixingoccurring within plugs moving through straight channels. By inducingtwirling, one may stimulate mixing; by preventing twirling, one maysuppress complete mixing. Suppressing mixing may be important in some ofthe reaction schemes, for example those shown in FIG. 5 and FIG. 6. Inthese reaction schemes, selective mixing of reagents A with reagent B,and also reagent C with reagent D, can occur without mixing of all fourreagents. Mixing of all four reagents occurs later as plugs movethrough, for example, the winding part of the channel. This approachallows several reactions to occur separated in time. In addition,suppressing mixing may be important when interfaces between plug fluidshave to be created, for example interfaces required for some methods ofprotein crystallization (FIG. 20).

The eddy at the tip of a developing plug may complicate visualizationand analysis of mixing. This eddy is normally significant in shortplugs, but only has a minor effect on long plugs. For applicationsinvolving visualization of mixing, the substrate is designed to includea narrow channel in the plug-forming region is designed such thatnarrow, elongated plugs form. Immediately downstream from theplug-forming region, the channel dimension is preferably expanded. Inthe expanded region of the channel(s), plugs will expand and becomeshort and rounded under the force of surface tension; this preserves thedistribution of the marker inside the plugs. This approach affords arelatively straightforward way of visualizing the mixing inside plugs ofvarious sizes. Video microscopy may be used to observe the distributionof colored markers inside the drops. A confocal microscope may also beused to visualize the average three-dimensional distribution of afluorescent marker. Visualization can be complemented or confirmed usinga Ca²⁺/Fluo-4⁻⁴ reaction. At millimolar concentrations, this reaction isexpected to occur with a half-life of about 1 μs. Thus, it can be usedto measure mixing that occurs on time scales of about 10 μs and longer.

The following discussion describes at least one method forthree-dimensional visualization of flows in plugs. Visualization ofchaotic transport in three-dimensions is a challenging task especiallyon a small scale. Predictions based on two-dimensional systems may beused to gain insight about plugs moving through a three-dimensionalmicrofluidic channel. Experiments and simulations involving atwo-dimensional system can aid in the design of channels that ensurechaotic flow in two-dimensional liquid plugs. Confocal microscopy hasbeen used to quantify steady, continuous three-dimensional flows inchannels. However, due to instrumental limitations of an opticalapparatus such as a confocal microscope, it is possible that the flowcannot be visualized with sufficiently high-resolution to observe, forexample, self-similar fractal structures characteristic of chaotic flow.Nonetheless, the overall dynamics of the flow may still be captured andthe absence of non-chaotic islands confirmed. Preferably, the channels(periodic or aperiodic) used in the visualization process are fabricatedusing soft lithography in PDMS. A PDMS replica is preferably sealedusing a thin glass cover slip to observe the flow using confocalmicroscopy.

In one experiment according to the invention, a series of line scans areused to obtain images of a three-dimensional distribution of fluorescentmarkers within the plugs. FIG. 10a ) is a schematic diagram depicting athree-dimensional confocal visualization of chaotic flows in plugs.Plugs are preferably formed from three laminar streams. The middlestream 11 preferably contains fluorescent markers. Preferably, themiddle stream 11 is injected into the channel system at a low volumetricflow rate. The volumetric flow rates of the two side streams 10, 12 arepreferably adjusted to position the marker stream in a desired sectionof the channel. Preferably, a confocal microscope such as a Carl ZeissLSM 510 is used. The LSM 510 is capable of line scans at about 0.38ms/512 pixel line or approximately 0.2 ms/100 pixel line. Fluorescentmicrospheres, preferably about 0.2 μm, and fluorescently labeledhigh-molecular weight polymers are preferably used to visualize the flowwith minimal interference from diffusion. A channel such as one with 100μm wide and 100 μm deep channel may be used. The line scan technique maybe applied to various sequences such as one that has about 200-μm longplugs separated by about 800-μm long oil stream.

A beam is preferably fixed in the x and z-directions and scannedrepeatedly back and forth along the y-direction. The movement of theplug in the x-direction preferably provides resolution along thex-direction. Line scan with 100 pixels across a 100 μm-wide channel willprovide a resolution of about 1 μm/pixel in the y-direction.Approximately 200 line scans per plug are preferably used to give aresolution of about 1 μm/pixel in the x-direction. For a 200 μm plugmoving at about 2000 μm/s, about 200 line scans are preferably obtainedover a period of about (200 μm)/(2000 μm/s)=0.1 s, or about 0.5 ms perline.

The sequence shown in FIG. 10b ) is preferably used for visualization ofa three dimensional chaotic flow. Each line scan preferably takes about0.2 ms with about 0.3 ms lag between the scans to allow the plug to moveby about 500 μm. Some optical distortions may result during theapproximately 0.2 ms scan as the plug is translated along thex-direction by about 0.2 μm. However, these distortions are believed tobe comparable to the resolution of the method. For a given positionalong the x-direction, a series of line scans are preferably obtainedfor about 10 seconds for each point along the z-direction to obtain anx-y cross-sections of ten plugs. Scans along the z-direction arepreferably taken in 1 μm increments to obtain a full three-dimensionalimage of the distribution of the fluorescent marker in the plug. Thisprocedure is preferably repeated at different positions along thex-direction to provide information such as changes in thethree-dimensional distribution of the fluorescent marker inside the plugas the plug moves along the channels.

In case of periodic perturbations, the fluorescent cross-sections of theplug in the y-z plane recovered from the above procedure representPoincaré sections corresponding to the evolution of the initial thinsheet of dye. The twirling of the aqueous phase upon formation of thesmall plugs could distribute the dye excessively throughout the plug andcould make visualization less conclusive. This twirling is preventedpreferably by designing a small neck in the plug-forming region, andthen beginning the first turn in a downward direction. This approach hasbeen successfully applied to flow visualization, and may be useful forconducting reactions.

Merging Plugs

The invention also provides a method of merging of plugs within asubstrate (see upper portion, FIG. 12(a)). Plugs are formed as describedabove. Plugs containing different reagents can be formed by separatelyintroducing different plug-fluids into a channel. The plugs containingdifferent reagents may be substantially similar in viscosity or maydiffer. The plugs containing different reagents may be substantiallysimilar in size or they may differ in size. Provided that the relativevelocities of the plugs containing different reagents differ, the plugswill merge in the channels. The location of merging can be controlled ina variety of ways, for example by varying the location of plug-fluidinlet ports, by varying the location of channel junctions (if one of theplug forming fluids is introduced into a secondary channel), varying thesize of the plugs, adjusting the speed at which different sets of plugsare transported varying the viscosity or surface tension of plugs havingsubstantially the same size, etc.

As shown in FIG. 12(a) (top photograph), plugs may be merged bydirecting or allowing the plugs 120, 121 to pass through a T-shapedchannel or a T-shaped region of a channel. The resulting merged plugs122 flow in separate channels or channel branches which may beperpendicular, as shown in FIG. 12(a), or nonperpendicular (FIG. 33).The merged plugs 122 may undergo further merging or undergo splitting,or they may be directed to other channels, channel branches, area, orregion of the substrate where they may undergo one or more reactions or“treatments” such as one or more types of characterizations,measurements, detection, sorting, or analysis.

In one embodiment, large and small plugs flow along separate channels orchannel branches towards a common channel where they merge. In a casewhere a large and a small plug do not converge at the same point at thesame time, they eventually form a merged plug as the larger plug, whichmoves faster than the smaller plug, catches up with the small plug andmerges with it. In the case where the larger and smaller plugs meet headon at the same point or region, they immediately combine to form amerged plug. The merged plugs may undergo splitting, described below, orfurther merging in other channels or channel regions, or they may bedirected to other channels, channel branches, area, or region of thesubstrate where they may undergo one or more types of characterizations,measurements, detection, sorting, or analysis.

In another embodiment, plugs can be merged by controlling the arrivaltime of the plugs flowing in opposite directions towards a common point,area, or region of the channel so that each pair of plugs arrive at thecommon point, area, or region of the channel at around the same time toform a single plug.

In another embodiment, an arched, semi-circular, or circular channelprovides a means for increasing the efficiency of plug merging. Thus,for example, a greater frequency of merging would occur within a morecompact area or region of the substrate. Using this scheme, plugsflowing along separate channels towards a common channel may mergewithin a shorter distance or a shorter period of time because thearched, semi-circular, or circular channel or channel branch converts orassists in converting initially out-of-phase plug pairs to in-phase plugpairs. Specifically, the arched, semi-circular, or circular channel orchannel branch would allow a lagging plug to catch up and merge with aplug ahead of it, thereby increasing the number of merged plugs in agiven period or a given area or region of a substrate.

Splitting and/or Sorting Plugs

The present invention also provides a method for splitting of plugswithin a substrate. Plugs can be split by passing a first portion of aplug into a second channel through an opening, wherein the secondchannel is downstream of where the plug is formed. Alternatively, plugsmay be split at a “Y” intersection in a channel. In both embodiment, theinitial plug splits into a first portion and a second portion andthereafter each portion passes into separate channel (or outlet). Eitherinitially formed plugs can be split or, alternatively, merged plugs canbe split. FIG. 6 shows a schematic diagram illustrating part of amicrofluidic network that uses multiple inlets (inlets 601, 603, 605,607 for reagents A, B, C, and D; inlets 602, 604, 606 for aqueousstreams) and that allows for both splitting and merging of plugs. Thisschematic diagram shows two reactions that are conducted simultaneously.A third reaction (between the first two reaction mixtures) is conductedusing precise time delay. Plugs can be split before or after a reactionhas occurred. In addition, FIG. 6 shows plugs at various stages ofmixing from the initial mixture 60 (A+B) and initial mixture 61 (C+D)through the mixed solutions 62 (A+B), 63 (C+D), and the 4-componentmixture 64 (A+B+C+D).

As shown in FIG. 12(b) (lower photograph), plugs may be split bydirecting or allowing the plugs 123, 124 to pass through a T-shapedchannel or a T-shaped region of a channel. In a preferred embodiment,the area or junction at which the plugs undergo splitting may benarrower or somewhat constricted relative to the diameter of the plugs acertain distance away from the junction. The resulting split plugs 125flow in separate channels or channel branches which may beperpendicular, as shown in FIG. 12(b), or nonperpendicular (FIG. 33).The split 125 plugs may undergo merging or further splitting, or theymay be directed to other channels, channel branches, area, or region ofthe substrate where they may undergo one or more reactions or“treatments” such as one or more types of characterizations,measurements, detection, sorting, or analysis.

In another embodiment, aqueous plugs can be split or sorted from an oilcarrier fluid by using divergent hydrophilic and hydrophobic channels.The channels are rendered hydrophilic or hydrophobic by pretreating achannel or region of a channel such that a channel or channel surfacebecomes predominantly hydrophilic or hydrophobic. As discussed in moredetail below, substrates with hydrophilic channel surfaces may befabricated using methods such as rapid prototyping inpolydimethylsiloxane. The channel surface can be rendered hydrophobiceither by silanization or heat treatment. For example,(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UnitedChemical Technologies, Inc.) vapor may be applied to the inlets of thesubstrate with dry nitrogen as a carrier gas to silanize the channelsurface.

Once plugs have been split into separate channels, further reactions canbe performed by merging the split plugs with other plugs containingfurther reactants.

Manipulation of plugs and reagents/products contained therein can alsobe accomplished in a fluid flow using methods or techniques such asdielectrophoresis. Dielectrophoresis is believed to produce movement ofdielectric objects, which have no net charge, but have regions that arepositively or negatively charged in relation to each other. Alternating,nonhomogeneous electric fields in the presence of plugs and/orparticles, cause the plugs and/or particles to become electricallypolarized and thus to experience dielectrophoretic forces. Depending onthe dielectric polarizability of the particles and the suspendingmedium, dielectric particles will move either toward the regions of highfield strength or low field strength. Using conventional semiconductortechnologies, electrodes can be fabricated onto a substrate to controlthe force fields in a micro fabricated device. Dielectrophoresis isparticularly suitable for moving objects that are electrical conductors.The use of AC current is preferred, to prevent permanent alignment ofions. Megahertz frequencies are suitable to provide a net alignment,attractive force, and motion over relatively long distances.

Radiation pressure can also be used in the invention to deflect and moveplugs and reagents/products contained therein with focused beams oflight such as lasers. Flow can also be obtained and controlled byproviding a thermal or pressure differential or gradient between one ormore channels of a substrate or in a method according to the invention.

Preferably, both the fluid comprising the plugs and the carrier fluidhave a relatively low Reynolds Number, for example 10⁻². The ReynoldsNumber represents an inverse relationship between the density andvelocity of a fluid and its viscosity in a channel of givencross-sectional dimension. More viscous, less dense, slower movingfluids will have a lower Reynolds Number, and are easier to divert,stop, start, or reverse without turbulence. Because of the small sizesand slow velocities, fabricated fluid systems are often in a lowReynolds number regime (R_(e)<<1). In this regime, inertial effects,which cause turbulence and secondary flows, are negligible and viscouseffects dominate the dynamics. These conditions are advantageous foranalysis, and are provided by devices according to the invention.Accordingly the devices according to the invention are preferablyoperated at a Reynolds number of less than 100, typically less than 50,preferably less than 10, more preferably less than 5, most preferablyless than 1.

Detection and Measurement

The systems of the present invention are well suited for performingoptical measurements using an apparatus such as a standard microscope.For example, PDMS is transparent in the visible region. When it is usedto construct a substrate, a glass or quartz cover slip can be used tocover or seal a PDMS network, thereby constructing a set of channelsthat can be characterized using visible, UV, or infrared light.Preferably, fluorescent measurements are performed, instead ofabsorption measurements, since the former has a higher sensitivity thanthe latter. When the plugs are being monitored by optical measurements,the refractive index of the carrier-fluid and the plug-fluids arepreferably substantially similar, but they can be different in certaincases.

In a plug-based system according to the invention, the relativeconcentrations (or changes in concentrations) can be typically measuredin a straightforward fashion. In some instances, the use of plugs toperform quantitative optical measurements of, for example, absoluteconcentrations is complicated by the presence of non-horizontaloil/water interfaces surrounding the plugs. These curved interfaces actas lenses, and may lead to losses of emitted light or opticaldistortions. Such distortions may adversely affect or prevent visualobservation of growing protein crystals, for example. Exact modeling ofthese losses is usually difficult because of the complicated shape thatthis interface may adopt at the front and back of a plug moving in anon-trivial pressure gradient.

This problem can be overcome or minimized in accordance with theinvention by using a technique such as refractive index matching. Thelosses and distortions depend on the difference between the refractiveindex (η_(D)) of the aqueous phase and the refractive index of theimmiscible carrier-fluid. Preferably, the carrier-fluid used in ananalysis have refractive indices that are substantially similar to thoseof water and aqueous buffers (TABLE 1), e.g., fluorinated oils havingrefractive indices near that of water close to the sodium D line at 589nm.

Preferably, for applications involving detection or measurement, thecarrier-fluids used are those having refractive indices that match thoseof commonly used aqueous solutions at the wavelengths used forobservation. To calibrate a system for quantitative fluorescencemeasurements, the plugs preferably contain known concentrations offluorescein. Preferably, the fluorescence originating from the plugs aremeasured and then compared with the fluorescence arising from the samesolution of fluorescein in the channel in the absence of oil. It isbelieved that when the refractive indexes are matched, the intensity (I)of fluorescence arising from the plugs will be substantially similar orequal to the intensity of the fluorescence from the aqueous solutionsafter making adjustments for the fraction of the aqueous stream:

I _(plug) =I _(solution) *V _(water)/(V _(water) +V _(oil))  Eqn. (3)

where V is the volumetric flow rate of the fluid streams. It is expectedthat smaller plugs with a higher proportion of curved interfaces willshow larger deviations from ideal plug behavior, i.e., those smallerplugs will tend to cause greater optical distortion. If necessary,measurements are performed partly to determine the errors associatedwith refractive index mismatch. Information from these measurements isuseful when unknown fluids are analyzed, or when a compromise betweenmatching the refractive index and matching the viscosities of the twofluids is required.

TABLE 1 Physical properties of some fluids used in certain embodimentsof the microfluidic devices. Refractive index, Viscosity, μ Fluid η_(D)[mPa-s] water 1.3330 1.00 aqueous PBS buffer, 1% 1.3343 1.02 aqueous PBSbuffer, 10% 1.3460 1.25 perfluorohexane 1.251 0.66perfluoro(methylcyclohexane) 1.30 1.56perfluoro(1,3-dimethylcyclohexane) 1.2895 1.92 perfluorodecaline 1.3145.10 perfluoroperhydrofluorene 1.3289 9.58 perfluoroperhydrophenanthrene1.3348 28.4 perfluorotoluene 1.3680 N/A hexafluorobenzene 1.3770 N/A

The detector can be any device or method for evaluating a physicalcharacteristic of a fluid as it passes through the detection region.Examples of suitable detectors include CCD detectors. A preferreddetector is an optical detector, such as a microscope, which may becoupled with a computer and/or other image processing or enhancementdevices to process images or information produced by the microscopeusing known techniques. For example, molecules can be analyzed and/orsorted by size or molecular weight. Reactions can be monitored bymeasuring the concentration of a product produced or the concentrationof a reactant remaining at a given time. Enzymes can be analyzed and/orsorted by the extent to which they catalyze a chemical reaction of anenzyme's substrate (conversely, an enzyme's substrate can be analyzed(e.g., sorted) based on the level of chemical reactivity catalyzed by anenzyme). Biological particles or molecules such as cells and virions canbe sorted according to whether they contain or produce a particularprotein, by using an optical detector to examine each cell or virion foran optical indication of the presence or amount of that protein. Achemical itself may be detectable, for example by a characteristicfluorescence, or it may be labeled or associated with a tag thatproduces a detectable signal when, for example, a desired protein ispresent, or is present in at least a threshold amount.

Practically any characteristic of a chemical can be identified ormeasured using the techniques according to the invention, provided thatthe characteristic or characteristics of interest for analysis can besufficiently identified and detected or measured to distinguishchemicals having the desired characteristic(s) from those which do not.For example, particulate size, hydrophobicity of the reagent versuscarrier-fluids, etc. can be used as a basis for analyzing (e.g., bysorting) plug-fluids, reaction products or plugs.

In a preferred embodiment, the plugs are analyzed based on the intensityof a signal from an optically detectable group, moiety, or compound(referred to here as “tag”) associated with them as they pass through adetection window or detection region in the device. Plugs having anamount or level of the tag at a selected threshold or within a selectedrange can be directed into a predetermined outlet or branch channel ofthe substrate. The tag signal may be collected by a microscope andmeasured by a detector such as a photomultiplier tube (PMT). A computeris preferably used to digitize the PMT signal and to control the flowthrough methods such as those based on valve action. Alternatively, thesignal can be recorded or quantified as a measure of the tag and/or itscorresponding characteristic or marker, e.g., for the purpose ofevaluation and without necessarily proceeding to, for example, sort theplugs.

In one embodiment according to the invention, a detector such as aphotodiode is larger in diameter than the width of the channel, forminga detection region that is longer (along the length of channel) than itis wide. The volume of such a detection region is approximately equal tothe cross sectional area of the channel above the diode multiplied bythe diameter of the diode.

To detect a chemical or tag, or to determine whether a chemical or taghas a desired characteristic, the detection region may include anapparatus (e.g., a light source such as a laser, laser diode, highintensity lamp such as mercury lamp) for stimulating a chemical or tagfor that characteristic to, for example, emit measurable light energy.In embodiments where a lamp is used, the channels are preferablyshielded from light in all regions except the detection region. Inembodiments where a laser is used, the laser can be set to scan across aset of detection regions. In addition, laser diodes may be fabricatedinto the same substrate that contains the analysis units. Alternatively,laser diodes may be incorporated into a second substrate (i.e., a laserdiode chip) that is placed adjacent to the analysis or sorter substratesuch that the laser light from the diodes shines on the detectionregion(s).

In preferred embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thedevice according to the invention. This design provides the advantagesof compactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing, for example, optical distortion.

As each plug passes into the detection region, it may be examined for acharacteristic or property, e.g., a corresponding signal produced by theplug, or the chemicals contained in the plugs, may be detected andmeasured to determine whether or not a given characteristic or propertyis present. The signal may correspond to a characteristic qualitativelyor quantitatively. Typically, the amount of signal corresponds to thedegree to which a characteristic is present. For example, the strengthof the signal may indicate the size of a molecule, the amount ofproducts(s) formed in a reaction, the amount of reactant(s) remaining,the potency or amount of an enzyme expressed by a cell, a positive ornegative reaction such as binding or hybridization of one molecule toanother, or a chemical reaction of a substrate catalyzed by an enzyme.In response to the signal, data can be collected and/or a flow controlcan be activated, for example, to direct a plug from one channel toanother. Thus, for example, chemicals present in a plug at a detectionregion may be sorted into an appropriate branch channel according to asignal produced by the corresponding examination at a detection region.Optical detection of molecular characteristics or the tag associatedwith a characteristic or property that is chosen for sorting, forexample, may be used. However, other detection techniques, for instanceelectrochemistry, or nuclear magnetic resonance, may also be employed.

In one embodiment according to the invention, a portion of a channelcorresponds to an analysis unit or detection region and includes adetector such as a photodiode preferably located in the floor or base ofthe channel. The detection region preferably encompasses a receive fieldof the photodiode in the channel, which receive field has a circularshape. The volume of the detection region is preferably the same as, orsubstantially similar, to the volume of a cylinder with a diameter equalto the receive field of the photodiode and a height equal to the depthof the channel above the photodiode.

The signals from the photodiodes may be transmitted to a processor viaone or more lines representing any form of electrical communication(including e.g. wires, conductive lines etched in the substrate, etc.).The processor preferably acts on the signals, for example by processingthem into values for comparison with a predetermined set of values foranalyzing the chemicals. In one embodiment, a value corresponds to anamount (e.g., intensity) of optically detectable signal emitted from achemical which is indicative of a particular type or characteristic of achemical giving rise to the signal. The processor preferably uses thisinformation (i.e., the values) to control active elements in adiscrimination region, for example to determine how to sort thechemicals (e.g., valve action).

When more than one detection region is used, detectors such asphotodiodes in a laser diode substrate are preferably spaced apartrelative to the spacing of the detection regions in the analysis unit.That is, for more accurate detection, the detectors are placed apart atthe same spacing as the spacing of the detection region.

A processor can be integrated into the same substrate that contains atleast one analysis unit, or it can be separate, e.g., an independentmicrochip connected to the analysis unit containing substrate viaelectronic leads that connect to the detection region(s) and/or to thediscrimination region(s), such as by a photodiode. The processor can bea computer or microprocessor, and is typically connected to a datastorage unit, such as computer memory, hard disk, or the like, and/or adata output unit, such as a display monitor, printer and/or plotter.

The types and numbers of chemicals based on the detection of, forexample, a tag associated with or bound to the chemical passing throughthe detection region, can be calculated or determined, and the dataobtained can be stored in the data storage unit. This information canthen be further processed or routed to a data outlet unit forpresentation, e.g. histograms representing, for example, levels of aprotein, saccharide, or some other characteristic of a cell surface inthe sample. The data can also be presented in real time as the sampleflows through a channel.

If desired, a substrate may contain a plurality of analysis units, i.e.,more than one detection region, and a plurality of branch channels thatare in fluid communication with and that branch out from thediscrimination regions. It will be appreciated that the position andfate of the reagents in the discrimination region can be monitored byadditional detection regions installed, for example, immediatelyupstream of the discrimination region and/or within the branch channelsimmediately downstream of the branch point. The information obtained bythe additional detection regions can be used by a processor tocontinuously revise estimates of the velocity of the reagents in thechannels and to confirm that molecules, particles, and substances havinga selected characteristic enter the desired branch channel.

In one embodiment, plugs are detected by running a continuous flowthrough a channel, taking a spatially resolved image with a CCD camera,and converting the relevant distance traversed by the plugs into time.

In another embodiment, plugs are detected following their exit through achannel point leading to a mass spectrometer (MS), e.g., an electrosprayMS. In this embodiment, time-resolved information (e.g., mass spectrum)can be obtained when the flow rate and the distance traversed by theplugs are known. This embodiment is preferable when one wants to avoidusing a label.

Varying the Concentration of Reagents Inside Plugs

The various devices and methods according to the invention allow thecontrol and manipulation of plug composition and properties. Forexample, they allow the variation of reagent concentration inside plugs.In one aspect according to the invention, the concentrations of thereagents in the plugs are varied by changing the relative flow rates ofthe plug-fluids. This is possible in conventional systems, but iscomplicated by problems of slow mixing and dispersion. Methods accordingto the invention are convenient for simultaneously testing a largenumber of experimental conditions (“screening”) because theconcentrations can be changed within a single setup. Thus, for example,syringes do not have to be disconnected or reconnected, and the inletsof a system according to the invention do not have to be refilled whenusing the above technique for varying the reagent concentrations inplugs.

The concentration of aqueous solutions inside plugs can be varied bychanging the flow rates of the plug-fluid streams (see FIGS.25(a)-25(c), discussed in detail in Example 11). In FIGS. 25(a)-25(c),water is introduced into inlets 251-258 at various flow rates whileperfluorodecaline flows through channels 259-261. In aqueous laminarflows, the ratio of flow rates of laminar streams in a microfluidicchannel may be varied from about 1000:1 and 1:1000, preferably 100:1 to1:100, more preferably 1:20 to 20:1.

The actual relative concentrations may be quantified using a solution ofknown concentration of fluorescein. In this example, the intensity of afluorescein stream can be used as a reference point to check forfluctuations of the intensity of the excitation lamp.

To illustrate an advantage offered by the invention over othertechniques, consider the following example. The method(s) described inthis example may be modified or incorporated for use in various types ofapplications, measurements, or experiments. Two or more reagents, suchas reagents A, B, C, are to be screened for the effects of differentconcentrations of reagents on some process, and the conditions underwhich an inhibitor can terminate the reaction of the enzyme with asubstrate at various enzyme and substrate concentrations is of interest.If A is an enzyme, B a substrate, and C an inhibitor, a substrate with 5inlets such as A/water/B/water/C inlets can be used, and the flow ratesat which A, B and C are pumped into the substrate can be varied.Preferably, the size of the plug is kept constant by keeping the totalflow rate of all plug-fluids constant. Because different amounts of A,B, C are introduced, the concentrations of A, B, C in the plugs willvary. The concentrations of the starting solutions need not be changedand one can rapidly screen all combinations of concentrations, as longas an enzymatic reaction or other reactions being screened can bedetected or monitored. Because the solutions are flowing and thetransport is linear, one can determine not only the presence or absenceof an interaction or reaction, but also measure the rate at which areaction occurs. Thus, both qualitative and quantitative data can beobtained. In accordance with the invention, the substrate typically neednot be cleaned between runs since most, if not all, reagents arecontained inside the plugs and leave little or no residue.

To extend the range over which concentrations can be varied, one may usea combination of, say, reagents A, B, C, D, E and prepare a micromolarsolution of A, a mM solution of B, and a M solution of C, and so on.This technique may be easier than controlling the flow rate over afactor of, say, more than 106. Using other known methods is likely to bemore difficult in this particular example because changing the ratio ofreagents inside the plug requires changing the size of the plugs, whichmakes merging complicated.

In another example, one may monitor RNA folding in a solution in thepresence of different concentrations of Mg²⁺ and H⁺. Previously, thiswas done using a stopped-flow technique, which is time consuming andrequires a relatively large amount of RNA. Using a method according tothe invention, an entire phase space can be covered in a relativelyshort period of time (e.g., approximately 15 minutes) using onlyμL/minute runs instead of the usual ml/shot runs.

These particular examples highlight the usefulness according to theinvention in, for example, the study of protein/protein interactionmediation by small molecules, protein/RNA/DNA interaction mediation bysmall molecules, or binding events involving a protein and several smallmolecules. Other interactions involving several components at differentconcentrations may also be studied using the method according to theinvention.

Generating Gradients in a Series of Plugs

In one aspect according to the invention, dispersion in apressure-driven flow is used to generate a gradient in a continuousstream of plug-fluid. By forming plugs, the gradient is “fixed”, i.e.,the plugs stop the dispersion responsible for the formation of thegradient. Although the stream does not have to be aqueous, an aqueousstream is used as a non-limiting example below.

FIGS. 44(a)-44(d) illustrate how an initial gradient may be created byinjecting a discrete aqueous sample of a reagent B into a flowing streamof water. In FIG. 44a ), the water+B mixture flowed through channel 441.Channels 443 and 445 contain substantially non-flowing water+B mixture.Water streams were introduced into inlets 440, 442, 444, 446-448 whileoil streams flowed through channels 449-452. FIG. 44d ) shows amultiple-inlet system through which reagents A, B, and C are introducedthrough inlets 453, 454, and 455. A pressure-driven flow is allowed todisperse the reagent along the channel, thus creating a gradient of Balong the channel. The gradient can be controlled by suitableadjustments or control of the channel dimensions, flow rates, injectionvolume, or frequency of sample or reagent addition in the case ofmultiple injections. This gradient is then “fixed” by the formation ofplugs. Several of these channels are preferably combined into a singleplug-forming region or section. In addition, complex gradients withseveral components may be created by controlling the streams. Thistechnique may be used for various types of analysis and synthesis. Forexample, this technique can be used to generate plugs for protein orlysozome crystallization. FIG. 42 shows an experiment involving theformation of gradients by varying the flow rates (the experimentaldetails are described in Example 17). FIG. 43 illustrates the use ofgradients to form lysozyme crystals (the experimental details aredescribed in Example 18).

Formation and Isolation of Unstable Intermediates

The devices and methods according to the present invention may also beused for synthesizing and isolating unstable intermediates. The unstableintermediates that are formed using a device according to the inventionare preferably made to undergo further reaction and/or analysis ordirected to other parts of the device where they may undergo furtherreaction and/or analysis. In one aspect, at least two differentplug-fluids, which together react to form an unstable intermediate, areused. As the unstable intermediates form along the flow path of thesubstrate, information regarding, for example, the reaction kinetics canbe obtained. Such unstable intermediates can be further reacted withanother reagent by merging plugs containing the unstable intermediatewith another plug-fluid. Examples of unstable intermediates include, butare not limited to, free radicals, organic ions, living ionic polymerchains, living organometallic polymer chains, living free radicalpolymer chains, partially folded proteins or other macromolecules,strained molecules, crystallization nuclei, seeds for compositenanoparticles, etc.

One application of devices according to the invention that involves theformation of unstable intermediates is high-throughput, biomolecularstructural characterization. It can be used in both a time-resolved modeand a non-time resolved mode. Unstable (and/or reactive) intermediates(for example hydroxyl radicals (OH)) can be generated in onemicrofluidic stream (for example using a known reaction of metal ionswith peroxides). These reactive species can be injected into anotherstream containing biomolecules, to induce reaction with thebiomolecules. The sites on the biomolecule where the reaction takesplace correlate with how accessible the sites are. This can be used toidentify the sites exposed to the solvent or buried in the interior ofthe biomolecule, or identify sites protected by another biomoleculebound to the first one. This method could be applied to understandingstructure in a range of biological problems. Examples include but arenot limited to protein folding, protein-protein interaction (proteinfootprinting), protein-RNA interaction, protein-DNA interactions, andformation of protein-protein complexes in the presence of a ligand orligands (such as a small molecule or another biomolecule). Interfacingsuch a system to a mass-spectrometer may provide a powerful method ofanalysis.

Experiments involving complex chemical systems can also be performed inaccordance with the invention. For example, several unstableintermediates can be prepared in separate plugs, such as partiallyfolded forms of proteins or RNA. The reactivity of the unstableintermediates can then be investigated when, for example, the plugsmerge.

Dynamic Control of Surface Chemistry

Control of surface chemistry is particularly important in microfluidicdevices because the surface-to-volume ratio increases as the dimensionsof the systems are reduced. In particular, surfaces that are generallyinert to the adsorption of proteins and cells are invaluable inmicrofluidics. Polyethylene glycols (PEG) and oligoethylene glycols(OEG) are known to reduce non-specific adsorption of proteins onsurfaces. Self-assembled monolayers of OEG-terminated alkane thiols ongold have been used as model substrates to demonstrate and carefullycharacterize resistance to protein adsorption. Surface chemistry towhich the solutions are exposed can be controlled by creatingself-assembled monolayers on surfaces of silicone or graftingPEG-containing polymers on PDMS and other materials used for fabricationof microfluidic devices. However, such surfaces may be difficult tomass-produce, and they may become unstable after fabrication, e.g.,during storage or use.

In one aspect according to the invention, the reagents inside aqueousplugs are exposed to the carrier-fluid/plug-fluid interface, rather thanto the device/plug-fluid interface. Using perfluorocarbons ascarrier-fluids in surface studies are attractive because they are insome cases more biocompatible than hydrocarbons or silicones. This isexemplified by the use of emulsified perfluorocarbons as bloodsubstitutes in humans during surgeries. Controlling and modifyingsurface chemistry to which the reagents are exposed can be achievedsimply by introducing appropriate surfactants into the fluorinated PFDphase.

In addition, the use of surfactants can be advantageous in problemsinvolving unwanted adsorption of substances or particles, for example,on the channel walls. Under certain circumstances or conditions, areaction may occur in one or more channels or regions of the substratethat give rise to particulates that then adhere to the walls of thechannels. When they collect in sufficient number, the adheringparticulates may thus lead or contribute to channel clogging orconstriction. Using methods according to the invention, such as usingone or more suitable surfactants, would prevent or minimize adhesion oradsorption of unwanted substances or particles to the channel wallsthereby eliminating or minimizing, for example, channel clogging orconstriction.

Encapsulated particulates may be more effectively prevented frominterfering with desired reactions in one or more channels of thesubstrate since the particulates would be prevented from directly cominginto contact with reagents outside the plugs containing theparticulates.

Fluorosurfactants terminated with OEG-groups have been shown todemonstrate biocompatibility in blood substitutes and other biomedicalapplications. Preferably, oil-soluble fluorosurfactants terminated witholigoethylene groups are used to create interfaces in the microfluidicdevices in certain applications. Surfactants with well-definedcomposition may be synthesized. This is preferably followed by thecharacterization of the formation of aqueous plugs in the presence ofthose surfactants. Their inertness towards nonspecific proteinadsorption will also be characterized. FIGS. 24(a)-24(c) show examplesof fluorinated surfactants that form monolayers that are: resistant toprotein adsorption; positively charged; and negatively charged. ForOEG-terminated surfactants, high values of n (≥16) are preferred formaking these surfactants oil-soluble and preventing them from enteringthe aqueous phase. In FIGS. 24(a)-24(c), compounds that have betweenabout 3 to 6 EG units attached to a thiol are sufficient to prevent theadsorption of proteins to a monolayer of thiols on gold, and are thuspreferred for inertness. In addition, surfactants that have been shownto be biocompatible in fluorocarbon blood substitutes may also be usedas additives to fluorinated carrier fluids.

Applications: Kinetic Measurements and Assays

The devices and methods of the invention can be also used for performingexperiments typically done in, for example, a microtiter plate where afew reagents are mixed at many concentrations and then monitored and/oranalyzed. This can be done, for example, by forming plugs with variablecomposition, stopping the flow if needed, and then monitoring the plugs.The assays may be positionally encoded, that is, the composition of theplug may be deduced from the position of the plug in the channel. Thedevices and methods of the invention may be used to performhigh-throughput screening and assays useful, for example, in diagnosticsand drug discovery. In particular, the devices and methods of theinvention can be used to perform relatively fast kinetic measurements.

The ability to perform fast measurements has revolutionized the field ofbiological dynamics. Examples include studies of protein C folding andcytochrome C folding. These measurements are performed using fastkinetics instruments that rely on turbulence to mix solutions rapidly.To achieve turbulence, the channels and the flow rates normally have tobe large, which require large sample volumes. Commercially availableinstruments for performing rapid kinetics studies can access times onthe order of 1 ms. The improved on-chip version of a capillaryglass-ball mixer gives a dead time of about 45 μs with a flow rate ofmore than about 0.35 mL/sec. The miniaturization of these existingmethods is generally limited by the requirement of high flow rate togenerate turbulence. Miniaturization afforded by devices and methodsaccording to the invention is advantageous because it allows, forexample, quantitative characterization, from genetic manipulation andtissue isolation, of a much wider range of biomolecules including thoseavailable only in minute quantities, e.g., microgram quantities. Inaddition, these new techniques and instruments afford a wide range ofaccessible time scales for measurements.

Time control is important in many chemical and biochemical processes.Typically, stopped-flow type instruments are used to measure reactionkinetics. These types of instruments typically rely on turbulent flow tomix the reagents and transport them while minimizing dispersion. Becauseturbulent flow occurs in tubes with relatively large diameters and athigh flow rates, stopped-flow instruments tend to use large volumes ofreagents (e.g., on the order of ml/s). A microfluidic analog of astopped-flow instrument that consumes small volumes of reagents, e.g.,on the order of μL/min, would be useful in various applications such asdiagnostics. Thus far, microfluidic devices have not been able tocompete with stopped-flow instruments because EOF is usually too slow(although it has less dispersion), and pressure-driven flows tend tosuffer from dispersion. In addition, mixing is usually very slow in bothsystems.

Stopped-flow instruments typically have sub-millisecond mixing, andcould be useful for experiments where such fast mixing is required. Thedevices and methods of the invention allow sub-millisecond measurementsas well. In particular, the present invention can be advantageous forreactions that occur on a sub-second but slower than about 1 or about 10millisecond (ms) time scale or where the primary concern is the solutevolume required to perform a measurement.

Further, if a plug is generated with two reactive components, it canserve as a microreactor as the plug is transported down a channel. Aplug's property, such as its optical property, can then be measured ormonitored as a function of distance from a given point or region of achannel or substrate. When the plugs are transported at a constant flowrate, a reaction time can be directly determined from a given distance.To probe the composition of the plug as it exits a channel, the contentsof the plugs may be injected into a mass spectrometer (e.g., anelectrospray mass spectrometer) from an end of the channel. The timecorresponding to the end of the channel may be varied by changing theflow rate. Multiple outlets may be designed along the channels to probe,for example, the plug contents using a mass spectrometer at multipledistance and time points.

An advantage of the devices and methods of the invention is that whenplugs are formed continuously, intrinsically slow methods of observationcan be used. For example, plugs flowing at a flow rate of about 10 cm/sthrough a distance of about 1 mm from a point of origin would be about10 ms old. In this case, the invention is particularly advantageousbecause it allows the use of a relatively slow detection method torepeatedly perform a measurement of, for example, 10 ms-old plugs forvirtually unlimited time. In contrast, to observe a reaction in astopped-flow experiment at a time, say, between about 9 and 11 ms, oneonly has about 2 ms to take data. Moreover, the present invention allowsone to obtain information involving complex reactions at several times,simultaneously, simply by observing the channels at different distancesfrom the point of origin.

The reaction time can be monitored at various points along achannel—each point will correspond to a different reaction or mixingtime. Given a constant fluid flow rate u, one may determine a reactiontime corresponding to the various times t₁, t₂, t₃, . . . t_(n) alongthe channel. Thus, if the distance between each pair of points n andn−1, which correspond to time tn and t_(n-1), are the same for a givenvalue of n, then the reaction time corresponding to point n along thechannel may be calculated from t_(n)=nl/u. Thus, one can convenientlyand repeatedly monitor a reaction at any given time t_(n). In principle,the substrate of the present invention allows one to cover a greatertime period for monitoring a reaction by simply extending the length ofthe channel that is to be monitored at a given flow rate or bydecreasing the flow rate over a given channel distance (see, forexample, FIGS. 22A-22D). In FIGS. 22A-22D, the following can beintroduced into the following inlets: enzyme into inlets 2201, 2205,2210, 2215; buffer into inlets 2202, 2206, 2211, 2216; substrate intoinlets 2203, 2207, 2212, 2217; buffer into inlets 2204, 2208, 2213,2218; inhibitor into inlets 2228, 2209, 2214, 2219. In FIGS. 22A-22D, acarrier fluid flows through the channel portions 2220, 2221, 2222, 2223from left to right. The channel portions enclosed by the dotted square2224, 2225, 2226, 2227 represent fields of view for the purpose ofmonitoring a reaction at various points along the channel.

The same principle applies to an alternate embodiment of the presentinvention, where the distance corresponding to a point n from a commonpoint of origin along the channel differs from that corresponding toanother channel by a power or multiples of 2. This can be seen moreclearly from the following discussion. Given a constant fluid flow rateu, one may determine a reaction time corresponding to the various timest₁, t₂, t₃, . . . t_(n) along the channel. Thus, if the distance betweeneach pair of points n and n−1, which correspond to time t_(n) andt_(n-1), are the same for a given value of n, then the reaction timecorresponding to point n along the channel may be calculated fromt_(n)=nl/u. In a relatively more complex channel geometry such as theone shown in FIG. 22(c), the corresponding equation is given byt_(n)=2^((n-1))l/u, which shows that the reaction times at variouspoints n varies as a power or multiples of 2.

In one aspect, channels according to the invention are used that placeinto a field of view different regions that correspond to different timepoints of a reaction. The channels according to the invention allowvarious measurements such as those of a complete reaction profile, aseries of linearly separated time points (such as those required for thedetermination of an initial reaction velocity in enzymology), and aseries of exponentially separated time points (e.g., first-order kineticmeasurements or other exponential analysis). Time scales in an imageframe can be varied from microseconds to seconds by, for example,changing the total flow rate and channel length.

FIG. 22A-D show various examples of geometries of microfluidic channelsaccording to the invention for obtaining kinetic information from singleoptical images. The illustrated channel systems are suitable for studiessuch as measurements of enzyme kinetics in the presence of inhibitors.The device shown in FIG. 22D has multiple outlets that can be closed oropened. In the device shown in FIG. 22D, preferably only one outlet isopen at a time. At the fastest flow rates, the top outlet is preferablyopen, providing reduced pressure for flow through a short fluid path 1.As flow rates are reduced, other outlets are preferably opened toprovide a longer path and a larger dynamic range for measurements at thesame total pressure.

In FIGS. 22B-22D, n is the number of segments for a given channel length1 traveled by the reaction mixture in time to (see p. 73, second fullparagraph for a related discussion of reaction times and channellengths). These systems allow the control of the ratio of reagents byvarying the flow rates. The systems also allow a quick quantification ofenzyme inhibition.

For example, ribonuclease A can be used with known inhibitors such asnucleoside complexes of vanadium and oxovanadium ions and other smallmolecules such as 5′-diphosphoadenosine 3′-phosphate and5′-diphosphoadenosine 2′-phosphate. The kinetics may be characterized byobtaining data and making Lineweaver-Burk, Eadie-Hofstee, or Hanes-Wolfeplots in an experiment. The experiment can be accomplished using only afew microliters of the protein and inhibitor solutions. This capabilityis particularly useful for characterizing new proteins and inhibitorsthat are available in only minute quantities, e.g., microgramquantities.

Kinetic measurements of reactions producing a fluorescent signal can beperformed according to the invention by analyzing a single imageobtained using, for example, an optical microscope. Long exposures(i.e., about 2 seconds) have been used to measure fast (i.e., about 2milliseconds) kinetics. This was possible because in a continuous flowsystem, time is simply equal to the distance divided by the flow rate.In the continuous flow regime in accordance with the invention, theaccessible time scales can be as slow as about 400 seconds, which can beextended to days or weeks if the flow is substantially slowed down orstopped. Typically, the time scale depends on the length of the channel(e.g., up to about 1 meter on a 3-inch diameter chip) at a low flow rateof about 1 mm/s, which is generally limited by the stability of thesyringe pumps, but may be improved using pressure pumping. The fastesttime scale is typically limited by the mixing time, but it may bereduced to about 20 μs in the present invention. Mixing time isgenerally limited by two main factors: (1) the mixing distance (e.g.,approximately 10-15 times the width of the channel); and (2) the flowrates (e.g., approximately 400 mm/s, depending on the capillary numberand the pressure drop required to drive the flow). Mixing distance isnormally almost independent of the flow rate. By using suitable designsof microfluidic channels, or networks of microfluidic channels, a widerange of kinetic experiments can be performed.

Reducing the channel size generally reduces the mixing time but it alsoincreases the pressure required to drive a flow. The equation belowdescribes the pressure drop, AP (in units of Pa), for a single-phaseflow in a rectangular capillary:

ΔP=28.42 Uμl/ab  Eqn. (9)

where U (m/s) is the velocity of the flow, μ (kilogram/meter-second, kgm⁻¹ s⁻¹) is the viscosity of the fluid, l (m) is the length of thecapillary, a (m) is the height of the capillary, and b (m) is the widthof the capillary. There is generally a physical limitation on how muchpressure a microfluidic device can withstand, e.g., about 3 atm for PDMSand about 5 atm for glass and Si. This limitation becomes crucial forvery small channels and restricts the total length of the channel andthus the dynamic range (the total distance through which this flow ratecan be maintained at a maximum pressure divided by the mixing distance)of the measurement.

FIG. 23 depicts a microfluidic network according to the invention withchannel heights of 15 and 2 μm. The channel design shown in FIG. 23illustrates how a dynamic range of about 100 can be achieved by changingthe cross-section of the channels. Under these conditions, mixing timein the winding channel is estimated to be about 25 μs and observationtime in the serpentine channels are estimated to be about 3 ms.

As FIG. 23 shows, rapid mixing occurs in the 2 μm×1 μm (height×width)channels and measurements are taken in the 2 μm×3 μm) channels. Thetable in FIG. 23 shows the distribution of the pressure drop, flowvelocity, and flow time as a function of the channel cross-sectiondimensions. A transition from a 1-μm wide to 3-μm wide channels shouldoccur smoothly, with plugs maintaining their stability and decreasingtheir velocity when they move from a 20-μm wide into a 50-μm widechannel. Changing the width of the channel can be easily done and easilyincorporated into a mask design. The height of the channel can bechanged by, for example, using photoresist layers having two differentheights that are sequentially spun on, for example, a silicon wafer. Atwo-step exposure method may then be used to obtain a microfluidicnetwork having the desired cross-section dimensions.

In another example of the application of the devices and methods of thepresent invention, the folding of RNase P catalytic domain (P RNAC-domain) of Bacillus subtilis ribozyme can be investigated usingchannels according to the invention. RNA folding is an important problemthat remains largely unsolved due to limitations in existing technology.Understanding the rate-limiting step in tertiary RNA folding isimportant in the design, modification, and elucidation of theevolutionary relationship of functional RNA structures.

The folding of P RNA C-domain is known to involve three populatedspecies: unfolded (U), intermediate (I), and native (N, folded) states.Within the first millisecond, the native secondary structure and some ofthe tertiary structure would have already folded (the RNA is compactedto about 90% of the native dimension) but this time regime cannot beresolved using conventional techniques such as stopped-flow. Usingchannels and substrates according to the invention, the time-dependenceof the P RNA folding kinetics upon the addition of Mg²⁺ can be studied.

Various types of assays (e.g., protein assays) known in the art,including absorbance assays, Lowry assays, Hartree-Lowry assays, Biuretassays, Bradford assays, BCA assays, etc., can be used, or suitablyadapted for use, in conjunction with the devices and methods of theinvention. Proteins in solution absorb ultraviolet light with absorbancemaxima at about 280 and 200 nm. Amino acids with aromatic rings are theprimary reason for the absorbance peak at 280 nm. Peptide bonds areprimarily responsible for the peak at 200 nm. Absorbance assays offerseveral advantages. Absorbance assays are fast and convenient since noadditional reagents or incubations are required. No protein standardneed be prepared. The assay does not consume the protein and therelationship of absorbance to protein concentration is linear. Further,the assay can be performed using only a UV spectrophotometer.

The Lowry assay is an often-cited general use protein assay. It was themethod of choice for accurate protein determination for cell fractions,chromatography fractions, enzyme preparations, and so on. Thebicinchoninic acid (BCA) assay is based on the same principle, but itcan be done in one step. However, the modified Lowry is done entirely atroom temperature. The Hartree version of the Lowry assay, a more recentmodification that uses fewer reagents, improves the sensitivity withsome proteins, is less likely to be incompatible with some saltsolutions, provides a more linear response, and is less likely to becomesaturated.

In the Hartree-Lowry assay, the divalent copper ion forms a complex withpeptide bonds under alkaline conditions in which it is reduced to amonovalent ion. Monovalent copper ion and the radical groups oftyrosine, tryptophan, and cysteine react with Folin reagent to producean unstable product that becomes reduced to molybdenum/tungsten blue. Inaddition to standard liquid handling supplies, the assay only requires aspectrophotometer with infrared lamp and filter. Glass or inexpensivepolystyrene cuvettes may be used.

The Biuret assay is similar in principle to that of the Lowry, howeverit involves a single incubation of 20 minutes. In the Biuret assay,under alkaline conditions, substances containing two or more peptidebonds form a purple complex with copper salts in the reagent. The Biuretassay offer advantages in that there are very few interfering agents(ammonium salts being one such agent), and there were fewer reporteddeviations than with the Lowry or ultraviolet absorption methods.However, the Biuret consumes much more material. The Biuret is a goodgeneral protein assay for batches of material for which yield is not aproblem. In addition to standard liquid handling supplies, a visiblelight spectrophotometer is needed, with maximum transmission in theregion of 450 nm. Glass or inexpensive polystyrene cuvettes may be used.

The Bradford assay is very fast and uses about the same amount ofprotein as the Lowry assay. It is fairly accurate and samples that areout of range can be retested within minutes. The Bradford is recommendedfor general use, especially for determining protein content of cellfractions and assessing protein concentrations for gel electrophoresis.Assay materials including color reagent, protein standard, andinstruction booklet are available from Bio-Rad Corporation. The assay isbased on the observation that the absorbance maximum for an acidicsolution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nmwhen binding to protein occurs. Both hydrophobic and ionic interactionsstabilize the anionic form of the dye, causing a visible color change.The assay is useful since the extinction coefficient of a dye-albumincomplex solution is constant over a 10-fold concentration range. Inaddition to standard liquid handling supplies, a visible lightspectrophotometer is needed, with maximum transmission in the region of595 nm, on the border of the visible spectrum (no special lamp or filterusually needed). Glass or polystyrene cuvettes may be used, but thecolor reagent stains both. Disposable cuvettes are recommended.

The bicinchoninic acid (BCA) assay is available in kit form from Pierce(Rockford, Ill.). This procedure is quite applicable to microtiter platemethods. The BCA is used for the same reasons the Lowry is used. The BCAassay is advantageous in that it requires a single step, and the colorreagent is stable under alkaline conditions. BCA reduces divalent copperion to the monovalent ion under alkaline conditions, as is accomplishedby the Folin reagent in the Lowry assay. The advantage of BCA is thatthe reagent is fairly stable under alkaline condition, and can beincluded in the copper solution to allow a one step procedure. Amolybdenum/tungsten blue product is produced as with the Lowry. Inaddition to standard liquid handling supplies, a visible lightspectrophotometer is needed with transmission set to 562 nm. Glass orinexpensive polystyrene cuvettes may be used.

The range of concentrations that can be measured using the above assaysrange from about 20 micrograms to 3 mg for absorbance at 280, betweenabout 1-100 micrograms for absorbance at 205 nm, between about 2-100micrograms for the Modified Lowry assay, between about 1-10 mg for theBiuret assay, between about 1-20 micrograms for the Bradford assay, andbetween about 0.2-50 micrograms for BCA assay. Many assays based onfluorescence or changes in fluorescence have been developed and could beperformed using methods and devices of the invention.

A detailed description of various physical and chemical assays isprovided in Remington: The Science and Practice of Pharmacy, A. R.Gennaro (ed.), Mack Publishing Company, chap. 29, “Analysis ofMedicinals,” pp. 437-490 (1995) and in references cited therein whilechapter 30 of the same reference provides a detailed description ofvarious biological assays. The assays described include titrimetricassays based on acid-base reactions, precipitation reactions, redoxreactions, and complexation reactions, spectrometric methods,electrochemical methods, chromatographic methods, and other methods suchas gasometric assays, assays involving volumetric measurements andmeasurements of optical rotation, specific gravity, and radioactivity.Other assays described include assays of enzyme-containing substances,proximate assays, alkaloidal drug assays, and biological tests such aspyrogen test, bacterial endotoxin test, depressor substances test, andbiological reactivity tests (in-vivo and in-vitro)

In addition, Remington: The Science and Practice of Pharmacy, A. R.Gennaro (ed.), Mack Publishing Company, chap. 31, “Clinical Analysis,”pp. 501-533 (1995) and references cited therein provide a detaileddescription of various methods of characterizations and quantitation ofblood and other body fluids. In particular, the reference includes adetailed description of various tests and assays involving various bodyfluid components such as erythrocytes, hemoglobin, thrombocyte,reticulocytes, blood glucose, nonprotein nitrogen compounds, enzymes,electrolytes, blood-volume and erythropoeitic mechanisms, and bloodcoagulation.

Nonlinear and Stochastic Sensing

Stochastic behavior has been observed in many important chemicalreactions, e.g., autocatalytic reactions such as inorganic chemicalreactions, combustion and explosions, and in polymerization ofsickle-cell hemoglobin that leads to sickle-cell anemia. Crystallizationmay also be considered an autocatalytic process. Several theoreticaltreatments of these reactions have been developed. These reactions tendto be highly sensitive to mixing.

Consider the extensively studied stochastic autocatalytic chemicalreaction between NaClO₂ and Na₂S₂O₃ (chlorite-thiosulfate reaction). Themechanism of this reaction can be described by reactions (1) and (2),

4S₂O₃2−+ClO₂ ⁻+4H⁺→2S₄O₆ ²⁻+2H₂O+Cl⁻ rate (v) α[H⁺]  (1)

S₂O₃ ²⁻+2ClO₂ ⁻+H₂O→2SO₄ ²⁻+2H⁺+2Cl⁻ rate (v) α[H⁺]²[C1]  (2)

where [H⁺] stands for the concentration of H. At a slightly basicpH=7.5, the slow reaction (1) dominates and maintains a basic pH of thereaction mixture (since the rate of this reaction v is directlyproportional [H⁺], this reaction consumes H⁺ and is auto-inhibitory).Reaction (2) dominates at acidic pH (since the rate of this reactionvaries in proportion to [H⁺]2[Cl⁻], this reaction produces both H⁺ andCl⁻ and is superautocatalytic). FIG. 21 shows the reaction diagram fortwo reactions corresponding to the curves 211, 212. The rates of the tworeactions (referred to here as reaction 211 and reaction 212) are equalat an unstable critical point at a certain pH. The lifetime of thereaction mixtures of NaClO₂ and NaS₂O₃ at this critical point cruciallydepends on stirring. In the absence of stirring, stochastic fluctuationsof [H⁺] in solution generate a localized increase in [H⁺]. This increasein [H⁺] marginally increases the rate of reaction 212, but it has a muchstronger accelerating effect on reaction 211 because of the higher-orderdependence on [H⁺] of this reaction. Therefore, in the region wherelocal fluctuations increase local [H⁺], reaction 211 becomes dominant,and more H⁺ is produced (which rapidly diffuses out of the region of theinitial fluctuation). The initiated chemical wave then triggers therapid reaction of the entire solution. Unstirred mixtures of NaClO₂ andNaS₂O₃ are stable only for a few seconds, and these fluctuations ariseeven in the presence of stirring.

FIG. 21 depicts a reaction diagram illustrating an unstable point in thechlorite-thiosulfate reaction. At [H⁺] values below the critical point,the slow reaction (1) dominates. At [H⁺] values above the criticalpoint, the autocatalytic reaction (2) dominates. The reaction mixture atthe [H⁺] value equal to the critical point is metastable in the absenceof fluctuations. Under perfect mixing, the effects of small fluctuationsaverage out and the system remains in a metastable state. Underimperfect mixing, fluctuations that reduce [H⁺] grow more slowly thanthose that increase [H⁺] due to the autocatalytic nature of reaction(2), and the reaction mixture thus rapidly becomes acidic.

It is known that chaotic flows should have a strong effect on diffusivetransport within the fluid (“anomalous diffusion”). It is also knownthat chaotic dynamics can lead to non-Gaussian transport properties(“strange kinetics”). In one aspect according to the invention, thesehighly unstable mixtures are stabilized in the presence of chaoticmixing using channels according to the invention because this mixing caneffectively suppress fluctuations. This invention can be used tounderstand the effects of mixing on the stochastic behavior of suchsystems, including for example, the chlorite thiosulfate system.

In a laminar flow, the flow profile in the middle of the channel is flatand there is virtually no convective mixing. Fluctuations involving [H⁺]that arise in the middle of the channel can grow and cause completedecomposition of the reaction mixture. Slow mixing reduces theprobability of fluctuations in plugs moving through straight channels.When fluctuations that occur in the centers of vortices are notefficiently mixed away, one or more spontaneous reactions involving someof the plugs can take place. In the present invention, chaotic mixing inplugs moving through winding channels efficiently mix out fluctuations,and thus substantially fewer or no spontaneous reactions are expected tooccur.

In a simple laminar flow, there is normally very little or no velocitygradient and substantially no mixing at the center of the channel. Thus,fluctuations that arise in the chlorite-thiosulfate reaction mixtureprepared at the critical [H⁺] are able to grow and lead to rapiddecomposition of the reaction mixture. Propagation of chemical fronts inautocatalytic reactions occurring in laminar flows has been describedwith numerical simulations, and back-propagation has been predicted(that is, a reaction front traveling upstream of the direction of thelaminar flow). Using the method of the present invention, thisback-propagation involving the reaction between NaClO₂ and NaS₂O₃ underlaminar flow conditions was observed.

In accordance with the invention, chaotic flow within plugs that flowthrough winding channels suppresses fluctuations and gives rise tostable reaction mixtures. There exists, of course, a finite probabilitythat fluctuations can arise even in a chaotically stirred plug. In oneaspect according to the invention, the details of the evolution of thesereactions are monitored using a high-speed digital camera. The plugs arepreferably separated by the oil and are not in communication with eachother, so the reaction of one plug will not affect the behavior of theneighboring plug. Statistics covering the behavior of thousands of plugscan be obtained quickly under substantially identical experimentalconditions.

Whether a fluctuation would be able to trigger an autocatalytic reactiondepends on factors such as the magnitude of a fluctuation and itslifetime. The lifetime of a fluctuation is typically limited by themixing time in the system. In an unstirred solution, mixing is bydiffusion and quite slow, and fluctuations may persist and lead toautocatalytic reactions. In a stirred solution, the lifetime of afluctuation is relatively short, and only large fluctuations havesufficient time to cause an autocatalytic reaction.

Mixing time and the lifetime of fluctuations typically depend on thesize of the plugs. As plug size decreases, mixing is accelerated andfluctuations are suppressed. However, very small plugs (e.g., about 1μm³ or 10⁻¹⁵ L) in a solution containing about 10⁻⁸ mole/literconcentration of H⁺ (pH=8) will contain only a few H⁺ ions per plug(about 10⁻²³ moles or about 6 H⁺ ions). When such small plugs areformed, the number of H⁺ ions in them will have a Poisson distribution.

An important experimental challenge is to establish that the stochasticbehavior in these systems is due mainly to internal fluctuations ofconcentrations. Other factors that may act as sources of noise andinstability are: (1) temporal fluctuations in the flow rates of theincoming reagent streams, which can lead to the formation of plugs withvarying amounts of reagents; (2) temperature fluctuations in solutionsin a microfluidic device, which may arise due to, for example,illumination by a microscope; and (3) fluctuations due to impurities incarrier-fluids leading to variations in the surface properties ofdifferent plugs.

Microfluidic systems according to the invention may be used to probevarious chemical and biochemical processes, such as those that showstochastic behavior in bulk due to their nonlinear kinetics. They canalso be used in investigating processes that occur in systems with verysmall volumes (e.g., about 1 μm³, which corresponds to the volume of abacterial cell). In systems with very small volumes, even simplereactions are expected to exhibit stochastic behavior due to the smallnumber of molecules localized in these volumes.

Autocatalytic reactions present an exciting opportunity for highlysensitive detection of minute amounts of autocatalysts. Several systemsare known to operate on this principle, silver-halide photography beingthe most widely used. In silver-halide photography, the energy ofphotons of light is used to decompose an emulsion of silver halide AgXinto nanometer-sized particles of metallic silver. A film that isembedded with the silver particles is then chemically amplified by theaddition of a metastable mixture of a soluble silver(I) salt and areducing agent (hydroquinone). Metallic silver particles catalyzereduction of silver(I) by hydroquinone, leading to the growth of theinitial silver particles. Another example of an autocatalytic reactionis the polymerase-chain reaction (PCR), which is a very effectiveamplification method that has been widely used in the biologicalsciences.

However, a dilemma occurs when designing systems with very highsensitivity and amplification. To achieve a very highly sensitiveamplification, the system typically has to be made very unstable. On theother hand, an unstable system is very sensitive to noise and has a veryshort lifetime. Also, in unstable systems, it is difficult todistinguish between spontaneous decomposition and a reaction caused bythe analyte. In one aspect, microfluidic devices according to theinvention, which allow chaotic mixing and compartmentalization, are usedto overcome this problem.

To demonstrate the potential of microfluidic systems according to thepresent invention, a microfluidic system according to the invention isused to handle unstable mixtures. In one application, a microfluidicsystem according to the invention is preferably used to control astochastic reaction between NaClO₂ and NaS₂O₃. In particular, thisreaction is preferably used for a highly sensitive amplificationprocess.

If a plug containing an unstable reaction mixture of NaClO₂ and NaS₂O₃is merged with a small plug containing an amount of H+ sufficient tobring the local concentration of H+ above critical, a rapidautocatalytic reactions is generally triggered. This autocatalyticreaction typically leads to the production of large amounts of H⁺. Thus,a weak chemical signal, e.g., a small amount of H⁺, is rapidly amplifiedby an unstable reaction mixture. Thus, for example, this approach can beused to investigate biological reactions such as those that involveenzymes, in which small amounts of H+ are produced.

The above autocatalytic system possesses several features thatcontribute to its novelty and usefulness. In one aspect, an unstableamplifying reaction mixture is prepared in-situ and is used withinmilliseconds before it has a chance to decompose. Preferably, the systemis compartmentalized so a reaction that occurs in one compartment doesnot affect a reaction in another compartment. This compartmentalizationallows thousands of independent experiments to be conducted in secondsusing only minute quantities of samples. Importantly, chaotic mixing inthe system reduces fluctuations and stabilizes the reaction mixture.

The applications of controlled autocatalytic amplification in accordancewith the invention are not limited to the detection of protons or Co²⁺ions. For example, the (Co(III)-5-Br-PAPS)/peroxomonosulfate oxidationreaction can also be used indirectly, for example, for a detection ofsmall amounts of peroxidase, which can be used as a labeling enzymebound to an antibody. The (Co(III)-5-Br-PAPS)/peroxomonosulfateoxidation reaction, which has been characterized analytically, involvesthe autocatalytic decomposition of violetbis[2-(5-bromo-pyridylazo)-5-(N-propyl-N-sulfopropyl-amino-phenolato]cobaltate,(Co(III)-5-Br-PAPS), upon oxidation with potassium peroxomonosulfate toproduce colorless Co²⁺ ions, which serve as the autocatalyst (the orderof autocatalysis has not been established for this reaction). (Endo etal., “Kinetic determination of trace cobalt(II) by visual autocatalyticindication,” Talanta, 1998, vol. 47, pp. 349-353; Endo et al.,“Autocatalytic decomposition of cobalt complexes as an indicator systemfor the determination of trace amounts of cobalt and effectors,”Analyst, 1996, vol. 121, pp. 391-394.)

Co(III)-[5-Br-PAPS]reduced+HSO₅ ⁻→Co²⁺+[5-Br-PAPS]oxidized+HSO⁴⁻

Addition of small amounts of Co²⁺ to the violet mixture of the(Co(III)-5-Br-PAPS and peroxomonosulfate produces an abrupt loss ofcolor to give a colorless solution. The time delay before thisdecomposition depends on the amount of the Co²⁺ added to the solution.This reaction has been used to detect concentrations of Co²⁺ as low as1×10⁻¹⁰ mole/L. The reaction shows good selectivity in the presence ofother ions (V(V), Cr(III), Cr(VI), Mn(II), Fe(II), Ni(II), Cu(II) andZn(II)).

The devices and methods according to the invention may be applied toother autocatalytic reactions, some of which have been described ininorganic, organic and biological chemistry. Reactions of transitionmetal ions such as Cr(III) (B82) Mn²⁺ or colloidal MnO₂, and reactionsof halides and oxohalides are often autocatalytic. Autocatalysisinvolving lanthanides (Eu²⁺) and actinides (U⁴⁺) has also been reported.All of these elements are potential targets for detection and monitoringin chemical waste, drinking water, or biological fluids. Intriguingpossibilities arise from using asymmetric autocatalytic reactions todetect minute amounts of optically active, chiral impurities, such asbiomolecules.

It is also possible to design new autocatalytic reactions. Autocatalysisis abundant in biology, and many enzymes are autocatalytic (e.g.,caspases involved in programmed cell death, kinases involved inregulation and amplification, and other enzymes participating inmetabolism, signal transduction, and blood coagulation. Emulsions ofperfluorocarbons such as perfluorodecaline (PFD) are used as bloodsubstitutes in humans during surgeries and should be compatible with avariety of biological molecules. Since the feasibility of quantitativemeasurements of enzyme kinetics has been demonstrated using plugs formedaccording to the invention, plugs formed according to the invention mayalso be applied to the detection of biological autocatalysts.

The devices and methods according to the present invention are notlimited to the detection of the autocatalyst itself. For example, thelabeling of an analyte using an autocatalyst is also within the scope ofthe present invention. Biomolecules are often labeled with metallicnanoparticles. Such metallic nanoparticles are highly effectiveautocatalysts for the reduction of metal ions to metals. Preferably, thesystems and methods of the present invention are used in the visualdetection of a single molecule of DNA, RNA, or protein labeled withnanoparticles via an autocatalytic pathway. In preliminary experimentsin accordance with the invention, clean particle formation and transportwithin plugs were observed.

In addition, the generation of metal (e.g., copper, silver, gold,nickel) deposits and nanoparticles upon chemical reduction also proceedby an autocatalytic mechanism. These reactions are commonly used forelectroless deposition of metals and should be useful for the detectionof minute amounts of metallic particles. The presence of metallicparticles in water can be indicative of the presence of operatingmechanical devices. In one aspect according to the invention, devicesand methods according to the invention are used to detect the presenceof minute or trace quantities of metallic particles.

The devices in accordance with the present invention are simple indesign, consume minute amounts of material, and robust. They do notrequire high voltage sources and can be operated, for example, usinggravity or a pocket-sized source of compressed air. In one aspect, thesystems according to the invention are used in portable and hand-helddevices.

Autocatalytic reactions show a threshold response, that is, there is avery abrupt temporal change from unreacted mixture to reacted mixture.In the case where time is equal to distance, this abrupt transition overa short distance can be observed using the devices and methods of theinvention. The time (and distance) is very sensitive to the initialconcentration of the catalyst, and thus it should be easy to determinethe concentration of the autocatalyst in the sample by noting how farthe reaction system traveled before it reacted.

One example of an autocatalytic process is blood coagulation. It is verysensitive to flow and mixing, therefore experimenting with it in theabsence of flow gives unreliable results or results that have littlerelevance to the real function of the coagulation cascade. A typicalmicrofluidic system may be difficult to use with blood because oncecoagulation occurs, it blocks the channel and stops the flow in themicrofluidic device. In addition, coagulated blood serves as anautocatalyst; even small amounts of coagulated blood in the channels canmake measurements unreliable.

These problems can be overcome using the devices of the presentinvention. Using plugs, autocatalytic reactions can be easilycontrolled, and the formation of solid clots would not be a problembecause any solids formed will be transported inside the plugs out ofthe channel without blocking the channel and without leavingautocatalytic residue. In addition, flow inside plugs can be easilycontrolled and adjusted to resemble flow under physiological conditions.

To address the sensitivity of blood coagulation to surfaces (the cascadeis normally initiated on the surface), microscopic beads containingimmobilized tissue factor (the cascade initiator) on the surface may beadded to one of the streams and transported inside the plugs. Also,surfactants may be used to control surface chemistry.

Thus, the devices and methods of the invention may be used, for example,to test how well the coagulation cascade functions (e.g., for hemophiliaor the tendency to form thrombus) under realistic flow conditions. Thistest would be particularly valuable in diagnostics. Blood may beinjected in one stream, and a known concentration of a molecule known toinduce coagulation (e.g., factor VIIa) can be added through anotherstream prior to plug formation. At a given flow rate, normal blood wouldcoagulate at a certain distance (which corresponds to a given time),which can be observed optically by light scattering or microscopy. Bloodof hemophiliac patients would coagulate at a later time. This type oftesting would be useful before surgical operations. In particular, thistype of testing is important for successful child delivery, especiallywhen hemophilia is suspected. Fetal testing may be performed since onlyminute amounts of blood are required by systems according to theinvention. The blood may be injected directly from the patient orcollected in the presence of anticoagulating agent (for example EDTA),and then reconstituted in the plug by adding Ca²⁺. In some cases, theaddition of Ca²⁺ may be sufficient to initiate the coagulation cascade.

The devices and methods of the invention may also be used to evaluatethe efficacy of anticoagulating agents under realistic flow conditions.Plugs can be formed from normal blood (which may be used directly orreconstituted by adding Ca²⁺ or other agents), an agent known to inducecoagulation, and an agent (or several agents that need to be compared)being tested as an anticoagulation agent. The concentrations of theseagents can be varied by varying the flow rates. The distance at whichcoagulation occurs is noted, and the efficacy of various agents toprevent coagulation is compared. The effects of flow conditions andpresence of various compounds in the system on the efficacy ofanticoagulation agents can be investigated quickly. The same techniquesmay also be used to evaluate agents that cause, rather prevent,coagulation. These tests could be invaluable in evaluating drugcandidates.

Synthesis

In accordance with the present invention, a method of conducting areaction within a substrate is provided. The reaction is initiated byintroducing two or more plug-fluids containing reactants into thesubstrate of the present invention.

In one aspect, the plug-fluids include a reagent and solvent such thatmixing of the plug-fluids results in the formation of a reactionproduct. In another embodiment, one of the plug-fluids may be reagentfree and simply contain fluid. In this embodiment, mixing of theplug-fluids will allow the concentration of the reagent in the plug tobe manipulated.

The reaction can be initiated by forming plugs from each plug-fluid andsubsequently merging these different plugs.

When plugs are merged to form merged plugs, the first and second set ofplugs may be substantially similar or different in size. Further, thefirst and second set of plugs may have different relative velocities. Inone embodiment, large arrays of microfluidic reactors are operated inparallel to provide substantial throughput.

The devices and methods of the invention can be used for synthesizingnanoparticles. Nanoparticles that are monodisperse are important assensors and electronic components but are difficult to synthesize(Trindade et al., Chem. Mat. 2001, vol. 13, pp. 3843-3858.). In oneaspect, monodisperse nanoparticles of semiconductors and noble metalsare synthesized under time control using channels according to theinvention (Park et al, J. Phys. Chem. B, 2001, vol. 105, pp.11630-11635.). Fast nucleation is preferably induced by rapid mixing,thereby allowing these nanoparticles to grow for a controlled period oftime. Then their growth is preferably quickly terminated by passivatingthe surfaces of the particles with, for example, a thiol. Nanoparticlesof different sizes are preferably obtained by varying the flow rate andtherefore the growth time. In addition, devices according to theinvention can be used to monitor the synthesis of nanoparticles, andthus obtain nanoparticles with the desired properties. For example, thenanoparticle formation may be monitored by measuring the changes in thecolor of luminescence or absorption of the nanoparticles. In addition,the growth of nanoparticles may be stopped by introducing a stream ofquenching reagent at a certain position along the main channel.

Rapid millisecond mixing generated in channels according to theinvention can help ensure the formation of smaller and much moremonodisperse nanoparticles than nanoparticles synthesized byconventional mixing of solutions. FIG. 13 shows the UV-VIS spectra ofCdS nanoparticles formed by rapid mixing in plugs (lighter shadespectrum with sharp absorption peak) and by conventional mixing ofsolutions (darker shade spectrum). The sharp absorption peak obtainedfor synthesis conducted in plugs indicates that the nanoparticles formedare highly monodisperse. In addition, the blue-shift (shift towardsshorter wavelengths) of the absorption peak indicates that the particlesformed are small.

FIGS. 14A(i)-A(iii) and 14B(i)-B(iii) illustrate the synthesis of CdSnanoparticles performed in PDMS microfluidic channels in single-phaseaqueous laminar flow (FIGS. 14A(i)-A(iii)) and in aqueous plugs thatwere surrounded by water-immiscible perfluorodecaline (FIGS.14B(i)-B(iii)). In FIGS. 14A(i)-A(iii) and 14B(i)-B(iii), Cd²⁺ wasintroduced into inlets 1400, 1403, aqueous stream was introduced intoinlets 1401, 1404, and S²⁻ was introduced into inlets 1402, 1405. InFIGS. 14A(i)-A(iii), an aqueous stream flowed through channel 1406 whilein FIG. 14B, oil flowed through channel 1407. FIGS. 14A(i)-A(iii) showportions of the channels 1408 and 1410 at time t=6 minutes and portionsof the channels 1409, 1411 at time t=30 minutes. It can be seen in FIGS.14A(i)-A(iii) that when laminar flow is used in the synthesis, largeamounts of CdS precipitate form on the channel walls. When plugs wereused for the synthesis, all CdS formed inside the plugs, and no surfacecontamination was observed. FIG. 15 illustrates a technique for thesynthesis of CdS nanoparticles, which is discussed in detail in Example13 below.

The following methods according to the invention can be used insynthesis involving nanoparticles:

(a) using self-assembled monolayers to nucleate nanoparticles withcrystal structures not accessible under homogeneous nucleationconditions (e.g., controlling polymorphism by controlling the surface atwhich nucleation takes place).

(b) using merging of plugs to create core-shell nanoparticles with arange of core and shell sizes. In a stream of plugs of a first channel,small core nanoparticles such as CdSe particles can be synthesized in amatter of few milliseconds. The CdSe particles can then be used as seedsfor mixing with solutions such as those containing Zn⁺² and S⁻². TheCdSe particles, acting as seeds for the formation of ZnS, thus allow theformation of CdSe(core)/ZnS(shell) nanoparticles. Core-shell particleswith more than two layers may be obtained by simply repeating themerging process more than once.

(c) using merging of plugs to create composite nanoparticles. Forexample, small nanoparticles of CdSe and ZnS can be formed using streamsof plugs from two separate channels. Merging of these streams leads toaggregation of these particles to form larger nanoparticles containingCdSe/ZnS composite. The composite nanoparticles that contain only a fewof the original nanoparticles can be made non-centrosymmetric, which mayhave interesting photophysical properties.

(d) using the devices and methods according to the invention tosynthesize medically important nanoparticles, such as encapsulated drugsand composite drugs.

(e) combinatorial synthesis of core-shell particles and other complexsystems. For example, the luminescence of CdSe/ZnS particles may bemonitored and the conditions adjusted to produce particles with variouscore and shell sizes, various doping impurities in the core and shell,and various ligand composition on the surface of the particles. Thesecan be conducted in real time using a device according to the invention.The entire process can also be automated.

The devices and methods according to the present invention may also beused for synthesizing polymers. Since the invention allows precisecontrol of the timing of a polymerization reaction, one or moreproperties of a polymer such as molecular weight, polydispersity andblockiness can be readily controlled or adjusted. In addition, use ofthe substrate of the present invention allows the user to precisely formblock copolymers by merging plugs within a device, since the path lengthof the channel will correspond to a specific duration of thepolymerization reaction. Similarly, a living polymer chain can beterminated with a specific end group to yield polymers with a discretesubset of molecular weights.

In addition, combinatorial libraries of drug candidates may besynthesized using similar approaches. The library may be encoded usingthe position of plugs in a channel. Plugs of variable composition may becreated by varying flow rates. Combination of synthesis of the librarymay be combined with screening and assays performed on the samemicrofluidic chip according to the present invention. In someembodiments, merging, splitting and sorting of plugs may be used duringsynthesis, assays, etc.

All of the above synthesis methods of the present invention can be usedto form macroscopic quantities of one or more reaction products byrunning multiple reactions in parallel.

Particle Separation/Sorting Using Plugs

The flow within the moving plugs can be used for separation of polymersand particles. Plugs can be used for separation by first using flowwithin a moving plug to establish a distribution of the polymers orparticles inside the plug (for example, an excess of the polymer insidethe front, back, right or left side of the plug) and then usingsplitting to separate and isolate the part of the plug containing higherconcentration of the polymers or particles. When two polymers orparticles are present inside the plug and establish differentdistributions, slitting can be used to separate the polymers orparticles. This approach may be useful, for example, in achieving on amicrofluidic chip any of, but not limited to, the following: separation,purification, concentration, membrane-less dialysis, and filtration.

Crystallization

The devices and methods of the invention allow fast, inexpensiveminiaturization of existing crystallization methods and other methodsthat can be adapted into, for example, novel protein screening andcrystallization techniques. The crystallization methods according to theinvention may be applied to various drugs, materials, small molecules,macromolecules, colloidal and nanoparticles, or any of theircombinations. Many relevant protein structures remain undetermined dueto their resistance to crystallization. Also, many interesting proteinsare only available in microgram quantities. Thus, a screening processmust permit the use of small amounts protein for analysis. Currentcrystallization screening technologies generally determine the idealconditions for protein crystallization on a milligram scale. Devices andmethods according to the invention improve current bench-top methodologyavailable to single users, and enables higher throughput automatedsystems with improved speed, sample economy, and entirely new methods ofcontrolling crystallization.

A microfluidic system according to the invention can be applied to thecrystallization of small molecules or macromolecules and theircomplexes.

For example, systems and methods in accordance with the presentinvention may include but are not limited to: (1) biologicalmacromolecules (cytosolic proteins, extracellular proteins, membraneproteins, DNA, RNA, and complex combinations thereof); (2) pre- andpost-translationally modified biological molecules (including but notlimited to, phosphorylated, sulfolated, glycosylated, ubiquitinated,etc. proteins, as well as halogenated, abasic, alkylated, etc. nucleicacids); (3) deliberately derivatized macromolecules, such as heavy-atomlabeled DNAs, RNAs, and proteins (and complexes thereof),selenomethionine-labeled proteins and nucleic acids (and complexesthereof), halogenated DNAs, RNAs, and proteins (and complexes thereof);(4) whole viruses or large cellular particles (such as the ribosome,replisome, spliceosome, tubulin filaments, actin filaments, chromosomes,etc.); (5) small-molecule compounds such as drugs, lead compounds,ligands, salts, and organic or metallo-organic compounds; (6)small-molecule/biological macromolecule complexes (e.g., drug/proteincomplexes, enzyme/substrate complexes, enzyme/product complexes,enzyme/regulator complexes, enzyme/inhibitor complexes, and combinationsthereof); (7) colloidal particles; and (8) nanoparticles.

Preferably, a general crystallization technique according to the presentinvention involves two primary screening steps: a crude screen ofcrystallization parameters using relatively small channels with a largenumber of small plugs, and a fine screen using larger channels andlarger plugs to obtain diffraction-quality crystals. For example, tencrude screens performed using channels with a (50 μm)² cross-sectionaldimension and with more or less one thousand 150-picoliter (pL) plugscorresponding to 10 mg/mL final concentration of a protein (10,000trials total) will typically require about 1.5 μL of solution, producecrystals up to about (10 μm)³ in size, and will consume approximately 15μg of protein. Up to 300 or more of such plugs can be formed in about 1second in these microfluidic networks. A fine screen around optimalconditions in (500 μm)² channels is expected to use more or less 50plugs. Another ˜5 μL of solution and another 50 μg of the protein areexpected to be consumed. This can produce crystals up to (100 μm)³ insize. Approximately 30 plugs can be formed about every second or so. Thethroughput of the system will generally be determined by the rate ofplug formation, and may be limited by how rapidly the flow rates can bevaried. Pressure control methods that operate at frequencies of 100 Hzare available and may be applied to PDMS microfluidic networks (Unger etal., “Monolithic fabricated valves and pumps by multilayer softlithography,” Science 2000, vol. 288, pp. 113-116.).

Crystal properties such as appearance, size, optical quality, anddiffractive properties may be characterized and measured under differentconditions. For example, a Raxis IIc X-ray detector mounted on a RigakuRU 200 rotating anode X-ray generator, which is equipped with doublefocusing mirrors and an MSC cryosystem, may be used for at least some ofthe characterizations and measurements. A synchrotron beam may be usefulfor characterization of small crystals. Also, these devices and methodsmay be used to build microfluidic systems according to the inventionthat are compatible with structural studies using x-ray beams.

A significant problem involving current crystallization approaches isdetermining the conditions for forming crystals with optimal diffractiveproperties. Normally crystals have to be grown, isolated, mounted, andtheir diffractive properties determined using an x-ray generator or asynchrotron. Microfluidic systems with thin, non-scattering walls wouldbe desirable for determining the diffractive properties of crystalsinside a microfluidic system. Preferably, crystallization is carried outinside this system using methods according to the invention, which aredescribed herein. The crystals are exposed to x-ray beams either todetermine their structure or diffractive properties (the screeningmode). For example, a PDMS membrane defining two side walls of thechannels could be sandwiched between two very thin glass plates(defining the top and bottom walls of the channels) that do notsignificantly scatter X-rays. Thus, the devices of the invention offer afurther advantage in that structural characterization could be conductedwhile the sample is inside the microfluidic device. Thus, the sample canbe characterized without the need to take out the sample, e.g., crystal,from the device.

The present system enables higher throughput automated systems withimproved speed, sample economy, and entirely new methods of controllingcrystallization. Microfluidic versions of microbatch, vapor phasediffusion and FID techniques may be carried out using the presentinvention, as described below, or using a combination of thesetechniques or other techniques. In addition, the nucleation and growthphases may be carried out in discrete steps through merging plugs, asdescribed herein.

Screening for protein crystallization involves varying a number ofparameters. During crystallization screening, a large number of chemicalcompounds may be employed. These compounds include salts, small andlarge molecular weight organic compounds, buffers, ligands,small-molecule agents, detergents, peptides, crosslinking agents, andderivatizing agents. Together, these chemicals can be used to vary theionic strength, pH, solute concentration, and target concentration inthe plug, and can even be used to modify the target. The desiredconcentration of these chemicals to achieve crystallization is variable,and can range from nanomolar to molar concentrations.

A typical crystallization mix contains set of fixed, butempirically-determined, types and concentrations of precipitation agent,buffers, salts, and other chemical additives (e.g., metal ions, salts,small molecular chemical additives, cryoprotectants, etc.). Water is akey solvent in many crystallization trials of biological targets, asmany of these molecules may require hydration to stay active and folded.Precipitation agents act to push targets from a soluble to insolublestate, and may work by volume exclusion, changing the dielectricconstant of the solvent, charge shielding, and molecular crowding.Precipitation agents compatible with the PDMS material of certainembodiments according to the invention include, but are not limited to,nonvolatile salts, high molecular weight polymers, polar solvents,aqueous solutions, high molecular weight alcohols, divalent metals.

Precipitation agents, which include large and small molecular weightorganics, as well as certain salts, may be used from under 1% to upwardsof 40% concentration, or from <0.5M to greater than 4M concentration.Water itself can act in a precipitating manner for samples that requirea certain level of ionic strength to stay soluble. Many precipitationagents may also be mixed with one another to increase the chemicaldiversity of the crystallization screen. Devices according to theinvention are readily compatible with a broad range of such compounds.

A nonexclusive list of salts that may be used as precipitation agents isas follows: tartrates (Li, Na, K, Na/K, NH₄); phosphates (Li, Na, K,Na/K, NH₄); acetates (Li, Na, K, Na/K, Mg, Ca, Zn, NH₄); formates (Li,Na, K, Na/K, Mg, NH₄); citrates (Li, Na, K, Na/K, NH₄); chlorides (Li,Na, K, Na/K, Mg, Ca, Zn, Mn, Cs, Rb, NH₄); sulfates (Li, Na, K, Na/K,NH₄); maleates (Li, Na, K, Na/K, NH₄); glutamates (Li, Na, K, Na/K,NH₄).

A nonexclusive list of organic materials that may be used asprecipitation agents is as follows: PEG 400; PEG 1000; PEG 1500; PEG 2K;PEG 3350; PEG 4K; PEG 6K; PEG 8K; PEG 10K; PEG 20K; PEG-MME 550; PEG-MME750; PEG-MME 2K; PEGMME 5K; PEG-DME 2K; dioxane; methanol; ethanol;2-butanol; n-butanol; t-butanol; jeffamine m-600; isopropanol;2-methyl-2,4-pentanediol; 1,6 hexanediol.

Solution pH can be varied by the inclusion of buffering agents; typicalpH ranges for biological materials lie anywhere between values of 3 and10.5 and the concentration of buffer generally lies between 0.01 and0.25 M. The microfluidics devices described in this document are readilycompatible with a broad range of pH values, particularly those suited tobiological targets.

A nonexclusive list of possible buffers that may be used according tothe invention is as follows: Na-acetate; HEPES; Na-cacodylate;Na-citrate; Na-succinate; Na—K-phosphate; TRIS; TRIS-maleate;imidazole-maleate; bistrispropane; CAPSO, CHAPS, MES, and imidazole.

Additives are small molecules that affect the solubility and/or activitybehavior of the target. Such compounds can speed up crystallizationscreening or produce alternate crystal forms or polymorphs of thetarget. Additives can take nearly any conceivable form of chemical, butare typically mono and polyvalent salts (inorganic or organic), enzymeligands (substrates, products, allosteric effectors), chemicalcrosslinking agents, detergents and/or lipids, heavy metals,organometallic compounds, trace amounts of precipitating agents, andsmall molecular weight organics.

The following is a nonexclusive list of additives that may be used inaccordance with the invention: 2-butanol; DMSO; hexanediol; ethanol;methanol; isopropanol; sodium fluoride; potassium fluoride; ammoniumfluoride; lithium chloride anhydrous; magnesium chloride hexahydrate;sodium chloride; calcium chloride dihydrate; potassium chloride;ammonium chloride; sodium iodide; potassium iodide; ammonium iodide;sodium thiocyanate; potassium thiocyanate; lithium nitrate; magnesiumnitrate hexahydrate; sodium nitrate; potassium nitrate; ammoniumnitrate; magnesium formate; sodium formate; potassium formate; ammoniumformate; lithium acetate dihydrate; magnesium acetate tetrahydrate; zincacetate dihydrate; sodium acetate trihydrate; calcium acetate hydrate;potassium acetate; ammonium acetate; lithium sulfate monohydrate;magnesium sulfate heptahydrate; sodium sulfate decahydrate; potassiumsulfate; ammonium sulfate; di-sodium tartrate dihydrate; potassiumsodium tartrate tetrahydrate; di-ammonium tartrate; sodium dihydrogenphosphate monohydrate; di-sodium hydrogen phosphate dihydrate; potassiumdihydrogen phosphate; di-potassium hydrogen phosphate; ammoniumdihydrogen phosphate; di-ammonium hydrogen phosphate; tri-lithiumcitrate tetrahydrate; tri-sodium citrate dihydrate; tri-potassiumcitrate monohydrate; diammonium hydrogen citrate; barium chloride;cadmium chloride dihydrate; cobaltous chloride dihydrate; cupricchloride dihydrate; strontium chloride hexahydrate; yttrium chloridehexahydrate; ethylene glycol; Glycerol anhydrous; 1,6 hexanediol; MPD;polyethylene glycol 400; trimethylamine HCl; guanidine HCl; urea;1,2,3-heptanetriol; benzamidine HCl; dioxane; ethanol; iso-propanol;methanol; sodium iodide; L-cysteine; EDTA sodium salt; NAD; ATP disodiumsalt; D(+)-glucose monohydrate; D(+)-sucrose; xylitol; spermidine;spermine tetra-HCl; 6-aminocaproic acid; 1,5-diaminopentane diHCl;1,6-diaminohexane; 1,8-diaminooctane; glycine; glycyl-glycyl-glycine;hexaminecobalt trichloride; taurine; betaine monohydrate;polyvinylpyrrolidone K15; non-detergent sulfo-betaine 195; non-detergentsulfo-betaine 201; phenol; DMSO; dextran sulfate sodium salt; JeffamineM-600; 2,5 Hexanediol; (+/−)-1,3 butanediol; polypropylene glycol P400;1,4 butanediol; tert-butanol; 1,3 propanediol; acetonitrile; gammabutyrolactone; propanol; ethyl acetate; acetone; dichloromethane;n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycolmonododecyl ether, nonaethylene glycol monolauryl ether; polyoxyethylene(9) ether; octaethylene glycol monododecyl ether, octaethylene glycolmonolauryl ether; polyoxyethylene (8) lauryl ether;Dodecyl-β-D-maltopyranoside; Lauric acid sucrose ester;Cyclohexyl-pentyl-β-D-maltoside; Nonaethylene glycol octylphenol ether;Cetyltrimethylammonium bromide;N,N-bis(3-D-gluconamidopropyl)-deoxycholamine;Decyl-β-D-maltopyranoside; Lauryldimethylamine oxide;Cyclohexyl-pentyl-β-D-maltoside; n-Dodecylsulfobetaine,3-(Dodecyldimethylanimonio)propane-1-sulfonate;Nonyl-β-D-glucopyranoside; Octyl-β-D-thioglucopyranoside, OS G;N,N-Dimethyldecylamine-β-oxide; Methyl0-(N-heptylcarbamoyl)-α-D-glucopyranoside; Sucrose monocaproylate;n-Octanoyl-β-D-fructofuranosyl-α-D-glucopyranoside;Heptyl-β-D-thioglucopyranoside; Octyl-β-D-glucopyranoside, OG;Cyclohexyl-propyl-β-D-maltoside;Cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine,3-(Decyldimethylammonio)propane-1sulfonate; Octanoyl-N-methylglucamide,OMEGA; Hexyl-β-D-glucopyranoside; Brij 35; Brij 58; Triton X-114; TritonX-305; Triton X-405; Tween 20; Tween 80; polyoxyethylene(6)decyl ether;polyoxyethylene(9)decyl ether; polyoxyethylene(10)dodecyl ether;polyoxyethylene(8)tridecyl ether; Decanoyl-N-hydroxyethylglucamide;Pentaethylene glycol monooctyl ether;3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate;3-[(3-Cholamidopropyl)-dimethylammonio]hydroxy-1-propane sulfonate;Cyclohexylpentanoyl-N-hydroxyethylglucamide;Nonanoyl-N-hydroxyethyglucamide;Cyclohexylpropanol-N-hydroxyethylglucamide;Octanoyl-N-hydroxyethylglucamide;Cyclohexylethanoyl-N-hydroxyethylglucamide; Benzyldimethyldodecylammonium bromide; n-Hexadecyl-β-D-maltopyranoside;n-Tetradecyl-β-D-maltopyranoside; n-Tridecyl-β-D-maltopyranoside;Dodecylpoly(ethyleneglycoether); n-Tetradecyl-N,N-dimethylammonio-1-propanesulfonate; n-Undecyl-β-D-maltopyranoside; n-DecylD-thiomaltopyranoside; n-dodecylphosphocholine; α-D-glucopyranoside,β-D-fructofuranosyl monodecanoate, sucrose mono-caprate;1-s-Nonyl-β-D-thioglucopyranoside; n-Nonyl-β-D-thiomaltoyranoside;N-Dodecyl-N,N-(dimethlammonio)butyrate; n-Nonyl-β-D-maltopyranoside;Cyclohexyl-butyl D-maltoside; n-Octyl-β-D-thiomaltopyranoside;n-Decylphosphocholine; n-Nonylphosphocholine;Nonanoyl-N-methylglucamide; 1-s-Heptyl-β-D-thioglucopyranoside;n-Octylphosphocholine; Cyclohexyl-ethyl D-maltoside;n-Octyl-N,N-dimethyl ammonio-1-propanesulfonate;Cyclohexyl-methyl-β-D-maltoside.

Cryosolvents are agents that stabilize a target crystal to flash-coolingin a cryogen such as liquid nitrogen, liquid propane, liquid ethane, orgaseous nitrogen or helium (all at approximately 100-120° K.) such thatcrystal becomes embedded in a vitreous glass rather than ice. Any numberof salts or small molecular weight organic compounds can be used as acryoprotectant, and typical ones include but are not limited to: MPD,PEG-400 (as well as both PEG derivatives and higher molecular-weight PEGcompounds), glycerol, sugars (xylitol, sorbitol, erythritol, sucrose,glucose, etc.), ethylene glycol, alcohols (both short- and long chain,both volatile and nonvolatile), LiOAc, LiCl, LiCHO₂, LiNO₃, Li2SO₄,Mg(OAc)₂, NaCl, NaCHO₂, NaNO₃, etc. Again, materials from whichmicrofluidics devices in accordance with the present invention arefabricated may be compatible with a range of such compounds.

Many of these chemicals can be obtained in predefined screening kitsfrom a variety of vendors, including but not limited to Hampton Researchof Laguna Niguel, Calif., Emerald Biostructures of Bainbridge Island,Wash., and Jena BioScience of Jena, Germany, that allow the researcherto perform both sparse matrix and grid screening experiments. Sparsematrix screens attempt to randomly sample as much of precipitant,buffer, and additive chemical space as possible with as few conditionsas possible. Grid screens typically consist of systematic variations oftwo or three parameters against one another (e.g., precipitantconcentration vs. pH). Both types of screens have been employed withsuccess in crystallization trials, and the majority of chemicals andchemical combinations used in these screens are compatible with the chipdesign and matrices in accordance with embodiments of the presentinvention. Moreover, current and future designs of microfluidic devicesmay enable flexible combinatorial screening of an array of differentchemicals against a particular target or set of targets, a process thatis difficult with either robotic or hand screening. This latter aspectis particularly important for optimizing initial successes generated byfirst-pass screens.

In addition to chemical variability, a host of other parameters can bevaried during crystallization screening. Such parameters include but arenot limited to: (1) volume of crystallization trial; (2) ratio of targetsolution to crystallization solution; (3) target concentration; (4)cocrystallization of the target with a secondary small or macromolecule;(5) hydration; (6) incubation time; (7) temperature; (8) pressure; (9)contact surfaces; (10) modifications to target molecules; and (11)gravity.

Although the discussion below refers to proteins, the particular devicesor methods described can also be used or suitably adapted for thecrystallization of other types of samples such as those mentioned above(e.g., small molecules, other macromolecules, nanoparticles, colloidalparticles, etc.). In one aspect of the present invention, proteincrystallization is conducted using miniaturized microbatch conditions.The process consists of two steps. First, plugs are preferably formedwherein the concentrations of the protein, precipitant, and additive areadjusted by varying the relative flow rates of these solutions. Thisstep corresponds to a screening step. Once the optimal concentrationshave been found, the flow rates can then be kept constant at the optimalconditions. In this step, plugs are preferably transported through thechannel as they form. Second, the flow is preferably stopped once thedesired number of plugs are formed. The plugs are then preferablyallowed to incubate. In some embodiments according to the invention theflow may be continued, rather than stopped. In those embodiments, theflow is maintained sufficiently slow and the channels are madesufficiently long that plugs spend sufficient time in the channels forcrystallization to occur (from tens of minutes to weeks, but may befaster or slower).

In one aspect, upon formation of the plugs, they are trapped usingexpansions in the channels. The expansions act as dead volume elementswhile the plugs are being formed in the presence of flow. Thus, theexpansions do not interfere with the flow of the plugs through thechannel. Once the flow is stopped, surface tension drives plugs into theexpansions where surface tension is minimized. The expansions may be,but are not limited to, oval, round, square, rectangular, orstar-shaped. In particular, a star-shaped expansion may preventadherence of the plug or of a crystal to the walls of the expansion. Theratio of the size of the expansion opening to the width of the channelmay be varied based on empirical results for a particular set ofconditions. FIG. 16 is a schematic illustration of a microfluidic deviceaccording to the invention that illustrates the trapping of plugs. Inexperiments, plugs were sustained in perfluorodecaline inside a channelfor one day, and did not appear to change during that time (a refractiveindex mismatch between the fluorinated and aqueous phase was introducedto aid in visualization of plugs).

The method described above allows a high degree of control over proteinand precipitant concentrations. It also allows a high degree of controlover a range of time scales through the control of plug size andcomposition. FIG. 17 shows a schematic of a microfluidic method forforming plugs with variable compositions for protein crystallization.Continuously varied flow rates of the incoming streams are preferablyused to form plugs with various concentrations of the protein,precipitation agents, and additives. In FIG. 17, for example, thefollowing can be introduced into the various inlets: buffers into inlets171, 172; PEG into inlet 173; salt into inlet 174; solvent into inlet175; and protein into inlet 176. These various solutions can enter achannel 177 through which a carrier fluid such as perfluorodecalineflows. For example, a 1-meter long channel with a 200×80 μm² crosssection can be used to form approximately two hundred 6 nL (nanoliter)plugs. If each plug contains enough protein to form a 40-μm³ crystal,200 trials will consume only about 1.2 μL of approximately 10 mg/mLprotein solution (12 μg of protein). About one minute may be sufficientto form plugs in these trials.

In another aspect according to the invention, after plugs are formed asdescribed above for the microbatch system, slow evaporation through avery thin PDMS membrane (or another membrane with slight waterpermeability) is preferably used for added control over thecrystallization process. A slow decrease in the volume of the plugduring evaporation is expected to produce a trajectory of the solutionthrough the crystallization phase space similar to that in a vapordiffusion experiment. Hence, this method, in addition to microbatchmethods, can be used to miniaturize and optimize vapor diffusionmethods.

In the vapor diffusion method, a drop containing protein, stabilizingbuffers, precipitants, and/or crystallization agents is allowed toequilibrate in a closed system with a much larger reservoir. Thereservoir usually contains the same chemicals minus the protein but atan over all higher concentration so that water preferentially evaporatesfrom the drop. If conditions are right, this will produce a gradualincrease in protein concentration such that a few crystals may form.

Vapor diffusion can be performed in two ways. The one most often used iscalled Hanging Drop Technique. The drop is placed on a glass coverslip,which is then inverted and used to seal a small reservoir in a LinbroPlate. After a period of several hours to weeks, microscopic crystalsmay form and continue to grow. The other set up is known as SittingDrop. In this method a drop (usually >10 uL) is placed in a depressionin either a Micro Bridge in a Linbro Plate or a glass plate and againplaced in a closed system to equilibrate with a much larger reservoir.One usually uses the sitting drop technique if the drop has very lowsurface tension, making it hard to turn upside down or if the drops needto be larger than 20 uL. Also, in some cases, crystals will grow betterusing one technique or the other.

In another embodiment, the plugs are preferably formed and transportedsuch that excessive mixing of the protein with the precipitation agentis minimized or prevented. For example, gentle mixing using spiralchannels may be used to achieve this and also to create interfacesbetween the protein and the precipitation agent. Alternatively,combining two streams of plugs in a T-junction without merging may beused to create plugs that diffuse and combine without significant mixingto establish a free interface after the flow is stopped. Diffusion ofthe proteins and precipitates through the interface inducescrystallization. This is an analogue of the Free-Interface Diffusionmethod. It may be performed under either the microbatch or vapordiffusion conditions as described above.

Preferably, the spacing between plugs can be increased or the oilcomposition changed to reduce plug-plug diffusion. For example, aspacing of about 2.5 mm in paraffin oil can be used, which has beenshown to be an effective barrier to aqueous diffusion in crystallizationtrials.

Visually identifying small crystals inside plugs with curved surfacescan be a challenge when performing microbatch experiments. In an aspectaccording to the invention, a method based on matching the refractiveindices of carrier-fluid with that of the plug fluid to enhancevisualization is used. Microscopic detection is preferably performed byusing shallow channels and by matching the refractive indices ofcarrier-fluid mixtures to those of the aqueous solutions.

In addition, at least three other novel methods of controlling proteincrystallization are described below: (1) using surface chemistry toeffect nucleation of protein crystals; (2) using different mixingmethods to effect crystallization; and (3) performing protein crystalsseeding by separating nucleation and growth phases in space.

Control of nucleation is one of the difficult steps in proteincrystallization. Heterogeneous nucleation is statistically a morefavorable process than its solution-phase counterpart. Ideal surfacesfor heterogeneous nucleation have complementary electrostatic maps withrespect to their macromolecular counterparts. Critical nuclei are morestable on such surfaces than in solution. Further, the degree ofsupersaturation required for heterogeneous nucleation is much less thanthat required for the formation of solution-phase nuclei. Surfaces suchas silicon, crystalline minerals, epoxide surfaces, polystyrene beads,and hair are known to influence the efficiency of proteincrystallization. Few studies have been done, but promising results havebeen shown for protein crystallization at the methyl, imidazole,hydroxyl, and carboxylic acid termini of self-assembled monolayers ongold. Using self-assembled monolayers, proteins were crystallized over abroader range of crystallization conditions and at faster rates thanwhen using the traditional silanized glass.

FIG. 18 is a schematic illustration of a method for controllingheterogeneous nucleation by varying the surface chemistry at theinterface of an aqueous plug-fluid and a carrier-fluid. In FIG. 18,plugs are formed in the presence of several solutions of surfactantsthat possess different functional groups (left side of the diagram). Theright side of FIG. 18 shows the aqueous phase region in which aprecipitant, solvent, and protein may be introduced into inlets 180,181, and 182, respectively. The composition of the surfactant monolayeris preferably controlled by varying the flow rates. In anotherapplication of the method illustrated in FIG. 18, the surface chemistrycan be varied continuously. The manipulation and control of the surfacechemistry can be used for screening, assays, crystallizations, and otherapplications where surface chemistry is important.

In one aspect of the invention, heterogeneous nucleation of proteins iscontrolled by forming aqueous plugs in a carrier-fluid, preferablycontaining fluoro-soluble surfactants if the carrier-fluid is afluorocarbon. Varying the relative flow rates of the surfactantsolutions may generate a wide variety of liquid-liquid interfaceconditions that can lead to the formation of mixed monolayers or mixedphase-separated monolayers. Preferably, several surfactants are used tocontrol the heterogeneous nucleation of protein crystals.Ethylene-glycol monolayers are preferably used to reduce heterogeneousnucleation, and monolayers with electrostatic properties complementaryto those of the protein are preferably used to enhance heterogeneousnucleation. These methods for controlling heterogeneous nucleation aredesigned to induce or enhance the formation of crystals that arenormally difficult to obtain. These methods may also be used to induceor enhance the formation of different crystal polymorphs that arerelatively more stable or better ordered.

As mentioned above, control of nucleation is highly desired in anadvanced crystallization screen. One method that can be used to achievecontrol of nucleation involves the transfer of nucleating crystals fromone concentration to another via dilution. This method, which has beenapplied in macroscopic systems primarily to vapor diffusion, wasintended to allow decoupling of the nucleation and growth phases. Thismethod is difficult to perform using traditional methods ofcrystallization because nucleation occurs long before the appearance ofmicrocrystals.

FIG. 19 illustrates a method of separating nucleation and growth using amicrofluidic network according to the present invention using proteinsas a non-limiting example. The left side of FIG. 19 shows plugs that areformed preferably using high concentrations of protein and precipitant.In FIG. 19, the following can be introduced into the various inletsshown: buffer into inlets 191, 196; PEG into inlets 192, 197;precipitant into inlets 193, 198; solvent into inlets 194, 199; andprotein into inlets 195, 200. Oil flows through the channels 201, 202from left to right. The portions 203, 204, and 205 of the channelcorrespond to regions where fast nucleation occurs (203), no nucleationoccurs (204), and where crystal growth occurs (205). The concentrationsused are those that correspond to the nucleating region in the phasediagram. Nucleation occurs as the plugs move through the channel to thejunction over a certain period. Preferably, these plugs are then mergedwith plugs containing a protein solution at a point corresponding to ametastable (growth, rather than nucleation) region (right side of FIG.19). This step ends nucleation and promotes crystal growth. When thecombined channel has been filled with merged plugs, the flow ispreferably stopped and the nuclei allowed to grow to produce crystals.

Nucleation time can be varied by varying the flow rate along thenucleation channel. The nucleus is preferably used as a seed crystal fora larger plug with solution concentrations that correspond to ametastable region. Existing data indicate the formation of nuclei withinless than about 5 minutes.

Fluid mixing is believed to exert an important effect in crystalnucleation and growth. Methods according to the invention are providedthat allow a precise and reproducible degree of control over mixing.FIG. 20 illustrates two of these methods. A method of mixing preferablyplaces the solution into a nucleation zone of the phase diagram withoutcausing precipitation. Preferably, gentle mixing (FIG. 20, left side) isused to achieve this by preventing, reducing, or minimizing contactbetween concentrated solutions of the protein and precipitant.Alternatively, rapid mixing (FIG. 20, right side) is used to achievethis by allowing passage through the precipitation zone sufficientlyquickly to cause nucleation but not precipitation. The two methods usedas examples involve the use of spiraling channels for gentle mixing andserpentine channels for rapid mixing.

The two methods in accordance with the invention depicted in FIG. 20 canbe used to determine the effect of mixing on protein crystallization. Inaddition, the various methods for controlling mixing describedpreviously (e.g., slow mixing in straight channels, chaotic mixing innon-straight channels, or mixing in which twirling may or may not occur)can be applied to crystallization, among other things.

After obtaining the crystals using any of the above describedtechniques, the crystals may be removed from the microfluidic device forstructure determination. In other systems, the fragile and gelatinousnature of protein crystals makes crystal collection difficult. Forexample, removing protein crystals from solid surfaces can damage themto the point of uselessness. The present invention offers a solution tothis problem by nucleating and growing crystals in liquid environments.In an aspect according to the invention, a thin wetting layer of acarrier-fluid covered with a surfactant is used to enable or facilitatethe separation of a growing crystal from a solid surface. When thecrystals form, they may be separated from the PDMS layer by using a thinlayer of a carrier-fluid.

It will be clear to one skilled in the art that while the abovetechniques are described in detail for the crystallization of proteins,techniques similar to the ones described above may also be used for thecrystallization of other substances, including other biomolecules orsynthetic chemicals. In addition, the devices and methods according tothe invention may be used to perform co-crystallization. For example, acrystal comprising more than one chemical may be obtained, for example,through the use of at least one stream of protein, a stream ofprecipitant, and optionally, a stream comprising a third chemical suchas an inhibitor, another protein, DNA, etc. One may then vary theconditions to determine those that are optimal for forming a co-crystal.

Particle Separation/Sorting Using Plugs

The flow within the moving plugs can be used for separation of polymersand particles. Plugs can be used for separation by first using flowwithin a moving plug to establish a distribution of the polymers orparticles inside the plug (for example, an excess of the polymer insidethe front, back, right or left side of the plug) and then usingsplitting to separate and isolate the part of the plug containing higherconcentration of the polymers or particles. When two polymers orparticles are present inside the plug and establish differentdistributions, splitting can be used to separate the polymers orparticles.

The invention is further described below, by way of the followingexamples. It will be appreciated by persons of ordinary skill in the artthat this example is one of many embodiments and is merely illustrative.In particular, the device and method described in this example(including the channel architectures, valves, switching and flow controldevices and methods) may be readily adapted, e.g., used in conjunctionwith one or more devices or methods, so that plugs may be analyzed,characterized, monitored, and/or sorted as desired by a user.

EXAMPLE Example 1—Fabrication of Microfluidic Devices and a GeneralExperimental Procedure

Microfluidic devices with hydrophilic channel surfaces were fabricatedusing rapid prototyping in polydimethylsiloxane. The channel surfaceswere rendered hydrophobic either by silanization or heat treatment. Tosilanize the surfaces of channels,(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UnitedChemical Technologies, Inc.) vapor was applied to the inlets of a devicewith dry nitrogen as a carrier gas at around 40-60 mm Hg above about 1atm pressure. Vacuum was simultaneously applied to the outlet of thedevice at about 650 mm Hg below atmospheric pressure. The silane vaporwas applied for a period of between about 1-3 hours. To treat thechannels using heat, a device was placed in an oven at approximately120° C. for about three hours. Alternatively, a device can be heated ina Panasonic “The Genius” 1300 Watt microwave oven at power set to “10”for about ten minutes.

Oils and aqueous solutions were pumped through devices using akdScientific syringe pump (Model 200) or Harvard Apparatus PhD 2000pump. Hamilton Company GASTIGHT syringes were used (10-250 μl) andHamilton Company 30 gauge Teflon® needles were used to attach thesyringes to the devices. Oils and aqueous solutions were pumped throughdevices at volumetric flow rates ranging from about 0.10 μL/min to about10.0 μL/min.

Aqueous solutions were colored using Crayola Original Formula Markers orFerroin Indicator (0.025 M, Fisher Scientific). Oils that were usedincluded perfluorodecaline (mixture of cis and trans, 95%, AcrosOrganics), perfluoroperhydrophenanthrene (tech., Alfa-Aesar), or1H,1H,2H,2H-perfluorooctanol (98%, Alfa-Aesar). The experiments weretypically performed using 10:1 mixtures of perfluorodecaline and1H,1H,2H,2H-perfluorooctanol.

The experiments were monitored using a Lica MZFLIII stereoscope withFostec (Schott-Fostec, LLC) Modulamps. Photographs of the experimentswere taken with a Spot Insight Color Camera, Model #3.2.0 (DiagnosticInstruments, Inc.). Spot Application version 3.4.0.0 was used to takethe photographs with the camera.

Example 2—Varying the Concentration of Aqueous Solutions in Plugs

The left side of each of FIGS. 25A-C shows a schematic diagram of themicrofluidic network and the experimental conditions. The right side ofeach of FIGS. 25A-C shows microphotographs illustrating the formation ofplugs using different concentrations of the aqueous streams. Aqueoussolutions of food dyes (red/dark and green/light) and water constitutedthe three streams. The volumetric flow rates of the three solutions(given in μL/min) are indicated. The dark stream is more viscous thanthe light stream. Therefore, the dark (more viscous) stream moves(measured in mm/s) more slowly and occupies a larger fraction of thechannel at a given volumetric flow rate.

FIG. 45a ) shows a schematic of the microfluidic network used todemonstrate that on-chip dilutions can be accomplished by varying theflow rates of the reagents. In FIG. 45a ), the reagents are introducedthrough inlets 451, 453 while the dilution buffer is introduced throughinlet 452. An oil stream flows through channel 454. The blue rectangleoutlines the field of view for images shown in FIG. 45c )-d). FIG. 45b )shows a graph quantifying this dilution method by measuring fluorescenceof a solution of fluorescein diluted in plugs in the microchannel. Dataare shown for 80 experiments in which fluorescein was flowed through oneof the three inlets, where C_(measured) and C_(theoretical) [μM] aremeasured and expected fluorescein concentration. FIG. 45(c) showsphotographs illustrating this dilution method with streams of food dyes455, 456, 457 having flow rates of 45 nL/s, 10 nL/s, and 10 nL/s,respectively. FIG. 45(d) shows photographs illustrating this dilutionmethod with streams of food dyes 458, 459, 460 having flow rates of 10nL/s, 45 nL/s, and 10 nL/s, respectively. Carrier fluid was flowed at 60nL/s.

Example 3

Networks of microchannels with rectangular cross-sections werefabricated using rapid prototyping in PDMS. The PDMS used was DowCorning Sylgard Brand 184 Silicone Elastomer, and devices were sealedusing a Plasma Prep II (SPI Supplies). The surfaces of the devices wererendered hydrophobic by baking the devices at 120° C. for 2-4 hours.

In FIGS. 26(a)-26(b), the red aqueous streams were McCormick® red foodcoloring (water, propylene glycol, FD&C Red 40 and 3, propylparaben),the green aqueous streams were McCormick® green food coloring (water,propylene glycol, FD&C yellow 5, FD&C blue 1, propylparaben) diluted 1:1with water, and the colorless streams were water. PFD used was a 10:1mixture of perfluorodecaline (mixture of cis and trans, 95%, AcrosOrganics):1H,1H,2H,2H-perfluorooctanol (Acros Organics). The red aqueousstreams were introduced in inlet 260, 265 (FIG. 26a )) while the greenaqueous streams were introduced in inlets 262, 263 in FIG. 26b ). Thecolorless aqueous stream was introduced in inlets 261, 264. The darkshadings of the streams and plug are due mainly from the red dye whilethe lighter shadings are due mainly from the green dye.

Aqueous solutions were pumped using 100 μL Hamilton Gastight syringes(1700 series, TLL) or 50 μL SGE gastight syringes. PFD was pumped using1 mL Hamilton Gastight syringes (1700 series, TLL). The syringes wereattached to microfluidic devices by means of Hamilton Teflon needles (30gauge, 1 hub). Syringe pumps from Harvard Apparatus (PHD 2000 Infusionpumps; specially-ordered bronze bushings were attached to the drivingmechanism to stabilize pumping) were used to infuse the aqueoussolutions and PFD.

Microphotographs were taken with a Leica MZ12.5 stereomicroscope and aSPOT Insight Color digital camera (Model #3.2.0, Diagnostic Instruments,Inc.). SPOT Advanced software (version 3.4.0 for Windows, DiagnosticInstruments, Inc.) was used to collect the images. Lighting was providedfrom a Machine Vision Strobe X-Strobe X1200 (20 Hz, 12 μF, 600V, PerkinElmer Optoelectronics). To obtain an image, the shutter of the camerawas opened for 1 second and the strobe light was flashed once with theduration of the flash being about 10 μs.

Images were analyzed using NIH Image software, Image J. Image J was usedto measure periods and lengths of plugs from microphotographs such asshown in FIG. 27b ). Periods corresponded to the distance from thecenter of one plug to the center of an adjacent plug, and the length ofa plug was the distance from the extreme front to the extreme back ofthe plug (see FIG. 28 for the definitions of front and back).Measurements were initially made in pixels, but could be converted toabsolute measurements by comparing them to a measurement in pixels ofthe 50 μm width of the channel.

To make measurements of the optical intensity of Fe(SCN)_(x) ^((3−x)+)complexes in plugs, microphotographs were converted from RGB to CMYKcolor mode in Adobe Photoshop 6.0. Using the same program, the yellowcolor channels of the microphotographs were then isolated and convertedto grayscale images, and the intensities of the grayscale images wereinverted. The yellow color channel was chosen to reduce the intensity ofbright reflections at the extremities of the plugs and at the interfacebetween the plugs and the channel. Following the work done in Photoshop,regions of plugs containing high concentrations of Fe(SCN)_(x) ^((3−x)+)complexes appeared white while regions of low concentration appearedblack. Using Image J, the intensity was measured across a thin,rectangular region of the plug, located halfway between the front andback of the plug (white dashed lines in FIG. 27a 1)). The camera used totake the microphotographs of the system was not capable of making linearmeasurements of optical density. Therefore, the measurements ofintensity were not quantitative. Several of the plots of intensityversus relative position across the channel (FIG. 27c ) were shiftedvertically by less than 50 units of intensity to adjust for non-uniformilluminations of different parts of the images. These adjustments werejustified because it was the shape of the distribution that was ofinterest, rather than the absolute concentration.

FIGS. 29a )-b) show plots of the sizes of periods and sizes of plugs asa function of total flow velocity (FIG. 29a )) and water fraction (wf)(FIG. 29b )). Values of capillary number (C.n.) were 0.0014, 0.0036,0.0072 and 0.011, while values of the Reynolds number (R_(e)) were 1.24,3.10, 6.21, and 9.31, each of the C.n. and R_(e) value corresponding toa set of data points with water fractions (wf) 0.20, 0.52, 0.52, and0.20 (the data points from top to bottom in FIG. 29A)). In turn, each ofthese sets of data points corresponds to a particular flow velocity asshown in FIG. 29a ). Plugs in FIG. 29b ) travel at about 50millimeter/second (mm/s). All measurements of length and size arerelative to the width of the channels (50 μm).

FIG. 30 shows microphotographs illustrating weak dependence of periods,length of plugs, and flow patterns inside plugs on total flow velocity.The left side of FIG. 30 shows a diagram of the microfluidic network.Here, the same solutions were used as in the experiment corresponding toFIGS. 27(a)(1)-(a)(2), 27(b), and 27(c)(1)-(c)(2). The Fe(SCN)_(x)^((3−x)+) solution was introduced into inlet 301 while the colorlessaqueous streams were introduced into inlets 302, 303. The same carrierfluid as used in the FIGS. 27(a)(1)-(a)(2), 27(b), and 27(c)(1)-(c)(2)experiment was flowed into channel 304. The right side of FIG. 30 showsmicrophotographs of plugs formed at the same water fraction (0.20), butat different total flow velocities (20, 50, 100, 150 mm/s from top tobottom). Capillary numbers were 0.0014, 0.0036, 0.0072, and 0.011,respectively, from top to bottom. Corresponding Reynolds numbers were1.24, 3.10, 6.21, and 9.31.

FIGS. 31A-C are plots showing the distribution of periods and lengths ofplugs where the water fractions were 0.20, 0.40, and 0.73, respectively.The total flow velocity was about 50 mm/s, C.n.=0.0036, R_(e)=3.10 inall cases.

FIGS. 27(a)(1)-(a)(2), 27(b), and 27(c)(1)-(c)(2) show the effects ofinitial conditions on mixing by recirculating flow inside plugs movingthrough straight microchannels. FIG. 27a 1) shows that recirculatingflow (shown by black arrows) efficiently mixed solutions of reagentsthat were initially localized in the front and back halves of the plug.Notations of front, back, left, and right are the same as that in FIG.28. FIG. 27a 2) shows that recirculating flow (shown by black arrows)did not efficiently mix solutions of reagents that were initiallylocalized in the left and right halves of the plugs. The left side ofFIG. 27b ) shows a schematic diagram of the microfluidic network. Thetwo colorless aqueous streams were introduced into inlets 271, 272 whilea carrier fluid in the form of perfluorodecaline flowed through channel273. These solutions did not perturb the flow patterns inside plugs.

The right side of FIG. 27b ) shows microphotographs of plugs of variouslengths near the plug-forming region of the microfluidic network forwater fractions of from 0.14 up to 1.00. FIG. 27c 1) shows a graph ofthe relative optical intensity of Fe(SCN)_(x) ^((3−x)+) complexes inplugs of varying lengths. The intensities were measured from left(x=1.0) to right (x=0.0) across the width of a plug (shown by whitedashed lines in FIG. 27a 1)-a2)) after the plug had traveled 4.4 timesits length through the straight microchannel. The gray shaded areasindicate the walls of the microchannel. FIG. 27c 2) is the same as FIG.27c 1) except that each plug had traversed a distance of 1.3 mm. The d/lof each water fraction (wf) were 15.2 (wf 0.14), 13.3 (wf 0.20), 11.7(wf 0.30), 9.7 (wf 0.40), 6.8 (wf 0.60), 4.6 (wf 0.73), and 2.7 (wf0.84), where d is the distance traveled by the plug and 1 is the lengthof the plug.

Example 4—Merging of Plugs

Experiments were conducted to investigate the merging of plugs usingdifferent channel junctions (T- or Y-shaped), cross-sections, and flowrates (see FIGS. 33a-d ). The figures on the left side of FIGS. 33a-dshow top views of microfluidic networks that comprise channels havingeither uniform or nonuniform dimension (e.g., the same or differentchannel diameters). The corresponding figures on the right aremicrophotographs that include a magnified view of two plug streams (fromthe two separate channels portions of which form the branches of theY-shaped junction) that merges into a common channel.

In FIG. 33a , the oil-to-water volumetric ratio was 4:1 in each pair ofoil and water inlets. The oil streams were introduced into inlets 330,332, while the aqueous streams were introduced into inlets 331, 333. Theflow rates of the combined oil/water stream past the junction where theoil and water meet was 8.6 mm/s. The channels, which were rectangular,had dimensions of 50 (width)×50 (height) μm². As shown in FIG. 33a ,plugs that flow in uniform-sized channels typically merged only whenthey simultaneously arrived at the T-junction. Thus, plug merging inthese channels occur infrequently. In addition, lagging plugs weretypically not able to catch up with leading plugs along the commonchannel.

FIG. 33b illustrates plug merging occurring between plugs arriving atdifferent times at the Y-shaped junction (magnified view shown). The oilstreams were introduced into inlets 334, 336, while the aqueous streamswere introduced into inlets 335, 337. In FIG. 33b , the flow rates forthe combined oil/water fluid past the junction where the oil and watermeet were 6.9 mm/s for channel 346 (the 50×50 μm² channel) and 8.6 mm/sfor channel 347 (the 25×50 μm² channel). The oil-to-water volumetricratio was 4:1 in each pair of oil and water inlets. The two channels(the branch channels) merged into a common channel 348 that had a 100×50μm² cross-section. As shown in the figure, the larger plugs from thebigger channel are able to merge with the smaller plugs from thenarrower channel even when they do not arrive at the junction at thesame time. This is because lagging larger plugs are able to catch upwith the leading smaller plugs once the plugs are in the common channel.

FIG. 33c depicts in-phase merging (i.e., plug merging upon simultaneousarrival of at least two plugs at a junction) of plugs of different sizesgenerated using different oil/water ratios at the two pairs of inlets.The oil streams were introduced into inlets 338, 340, while the aqueousstreams were introduced into inlets 339, 341. The flow ratecorresponding to the fluid stream through channel 349 resulting from a1:1 oil-to-water volumetric ratio was 4.0 mm/s, while that throughchannel 350 corresponding to the 4:1 oil-to-water volumetric ratio was6.9 mm/s. Each branch channel of the Y-shaped portion of the network(magnified view shown) had a dimension of 50×50 μm² while the commonchannel 351 (the channel to which the branch channels merge) was 125×50μm².

FIG. 33d illustrates defects (i.e., plugs that fail to undergo mergingwhen they would normally merge under typical or ideal conditions)produced by fluctuations in the relative velocity of the two incomingstreams of plugs. The oil streams were introduced into inlets 342, 344,while the aqueous streams were introduced into inlets 343, 345. In thisexperiment, the flow rate corresponding to the fluid stream throughchannel 352 resulting from a 1:1 oil-to-water volumetric ratio was 4.0mm/s, while that through channel 353 corresponding to the 4:1oil-to-water volumetric ratio was 6.9 mm/s. Each branch channel thatformed one of the two branches of the Y-shaped intersection (magnifiedview shown) was 50×50 μm² while the common channel 354 (the channel towhich the two branch channels merge) is 125×50 μm².

Example 5—Splitting Plugs Using a Constricted Junction

The splitting of plugs was investigated using a channel network with aconstricted junction. In this case, the plugs split and flowed past thejunction into two separate branch channels (in this case, branchchannels are the channels to which a junction branches out) that are ata 180°-angle to each other (see FIGS. 34a-c each of which show a channelnetwork viewed from the top). In these experiments, the outletpressures, P₁ and P₂, past the constricted junction were varied suchthat either P1≈P2 (FIG. 34b ) or P₁<P₂ (FIG. 34c ). Here, the relativepressures were varied by adjusting the relative heights of the channelsthat were under pressures P₁ and P₂. Since longer plugs tend to splitmore reliably, this branching point (or junction) was made narrower thanthe channel to elongate the plugs. FIG. 34a shows a schematic diagram ofthe channel network used in the experiment. The oil and water wereintroduced into inlets 3400 and 3401, respectively. The oil-to-waterratio was 4:1 while the flow rate past the junction where the oil andwater meet was 4.3 mm/s.

FIG. 34b is a microphotograph showing the splitting of plugs into plugsof approximately one-half the size of the initial plugs. The channels3404, which were rectangular, had a cross-section that measured 50×50μm². The constricted section of the channel 3402 right next to thebranching point measured 25×50 μm². The outlet pressures, P₁ and P₂,were about the same in both branch channels. Here, the plugs split intoplugs of approximately the same sizes.

FIG. 34c is a microphotograph showing the asymmetric splitting of plugs(i.e., the splitting of plugs into plugs of different sizes or lengths)which occurred when P₁<P₂. The microphotograph shows that larger plugs(somewhat rectangular in shape) flowed along the channel with the lowerpressure P₁; while smaller plugs (spherical in shape) flowed along thechannel with the higher pressure P₂. As in FIG. 34b , each of thechannel 3405 cross-section measured 50×50 μm². The constricted sectionof the channel 3403 at the junction measured 25×50 μm².

Example 6—Splitting Plugs without Using a Constricted Junction

The splitting of plugs was investigated using a channel network withouta constriction such as the one shown in FIGS. 35b-c . The channelnetwork used was similar to that shown in FIG. 34(a) except that herethe plugs split and flowed past the junction in two separate channels ata 90°-angle to each other (the plug flow being represented by arrows).The oil and aqueous streams (4:1 oil:aqueous stream ratio) wereintroduced into inlets 3500 and 3501, respectively. An oil-only streamflowed through channel 3502. All channels had a cross-section of 50×50μm². The flow rate used was 4.3 mm/s. FIGS. 35a-c , which represent topviews of a channel network, show that plugs behave differently comparedto the plugs in Example 3 when they flow past a junction in the absenceof a channel constriction, such as a constriction shown in FIGS. 35b-c .As FIG. 35c shows, when P₁<P₂, the plugs remained intact after passingthrough the junction. Further, the plugs traveled along the channel thathad the lower pressure (P₁ in FIG. 35c ) while the intervening oilstream split at the junction. The splitting of the oil stream at thejunction gives rise to a shorter separation between plugs flowing alongthe channel with pressure P₁ compared to the separation between plugs inthe channel upstream of the branching point or junction.

Example 7—Monitoring Autocatalytic Reactions Using a Microfluidic System

FIG. 37 illustrates the design of an experiment involving chemicalamplification in microfluidic devices according to the invention thatinvolves an investigation of a stochastic autocatalytic reaction. Thisexample illustrates how the devices of the present invention can be usedto study the acid-sensitive autocatalytic reaction between NaClO₂ andNaS₂O₃. On the left side of the microfluidic network, a three-channelinlet introduces an aqueous stream through channel 3702, an esterthrough channel 3701, and an esterase through channel 3703. Oil flowedthrough channels 3713, 3714. The reaction between ester and esteraseyield plugs 3704 that contain a small amount of acid. On the right sideof the microfluidic network, the five-channel inlet introduces NaClO₂through inlet 3705, an aqueous stream through inlet 3706, a pH indicatorthrough inlet 3707, a second aqueous stream through inlet 3708, andNaS2O3 through channel 3709. A carrier fluid flows through channels3713, 3714. Unstirred mixtures of NaClO₂ and NaS₂O₃ are highly unstableand even a slight concentration fluctuation within that mixture leads torapid decomposition. Thus, the plugs 3710 containing NaClO₂/NaS₂O₃mixture must not only be quickly mixed but also promptly used afterformation. In this proposed experiment, the curvy channels promotechaotic mixing. When a slightly acidic plug of the ester-esterasereaction is merged with a plug of an unstable NaClO₂/NaS₂O₃ mixture atthe contact region 3712, an autocatalytic reaction will generally betriggered. Upon rapid mixing of these two plugs, the resulting plugs3711 become strongly acidic. The pH indicator introduced in thefive-channel inlet is used to visualize this entire amplificationprocess.

Example 8—Using Chemical Reactions as Highly Sensitive AutoamplifyingDetection Elements in Microfluidic Devices

In one aspect according to the invention, a sequential amplificationusing controlled autocatalytic systems is used to amplify samples thatcontain single molecules of autocatalysts into samples containing asufficiently high concentration of an autocatalyst such that theamplified autocatalyst can be detected with the naked eye can bedetected with the naked eye. Although systems displaying stochasticbehavior are expected to display high sensitivity and amplification,various autocatalytic systems can be used in accordance with theinvention. A sequential amplification using the microfluidic devicesaccording to the invention can be illustrated using a reaction that hasbeen characterized analytically: the autocatalytic decomposition ofvioletbis[2-(5-bromo-pyridylazo)-5-(N-propyl-N-sulfopropyl-amino-phenolato]cobaltate,(Co(III)-5-Br-PAPS), upon oxidation with potassium peroxomonosulfate toproduce colorless Co²⁺ ions. Here, the Co²⁺ ions serve as theautocatalyst (the order of autocatalysis, m, has not been establishedfor this reaction).

Co(III)-[5-Br-PAPS]reduced+HSO₅ ⁻→Co²⁺+[5-Br-PAPS]oxidized+HSO⁴⁻  (3)

Addition of small amounts of Co²⁺ to the violet mixture of(Co(III)-5-Br-PAPS and peroxomonosulfate produces an abrupt loss ofcolor to give a colorless solution. The time delay before thisdecomposition depends on the amount of the Co²⁺ added to the solution.This reaction has been used to detect concentrations of Co²⁺ as low asabout 1×10⁻¹⁰ mole/L. The reaction shows good selectivity in thepresence of other ions (V(V), Cr(III), Cr(VI), Mn(II), Fe(II), Ni(II),Cu(II) and Zn(II)).

To use this reaction for amplification, a microfluidic network as shownin FIG. 38 is preferably used. An unstable solution ofCo(III)-[5-Br-PAPS]reduced and peroxomonosulfate at pH=7 buffer in largeplugs are preferably formed in a channel. These large plugs arepreferably split in accordance with the invention into three differentsizes of plugs. Preferably, the plug sizes are (1 μm)³=10⁻¹⁵ L in thefirst channel; (10 μm)³=10⁻¹² L in the second channel; and (100μm)³=10⁻⁹ L in the third channel. A three-step photolithography ispreferably used in the fabrication of masters for these microfluidicchannels.

Example 9—Multi-Stage Chemical Amplification in Microfluidic Devices forSingle Molecule Detection FIG. 38 illustrates a method for a multi-stagechemical amplification for single molecule detection using microfluidicdevices according to the invention. This example illustrates the use ofan autocatalytic reaction between Co(III)-5-Br-PAPS (introduced throughinlet 3803) and KHSOS (introduced through inlet 3801) in a pH=7 buffer(introduced through inlet 3802) that is autocatalyzed by Co²⁺ ions. Oilstreams are allowed to flow through channels 3804, 3805. This reactionmixture (contained in plugs 3811) is unstable and decomposes rapidly(shown in red) when small amounts of Co²⁺ 3810 are added. Thus, thisreaction mixture is preferably mixed quickly and used immediately. Thereaction mixture is preferably transported through the network in (1μm)³, (10 μm)³, (100 μm)³ size plugs. On the left side of themicrofluidic network, the approximately 1 μm³ plugs of the sample to beanalyzed form at a junction of two channels (shown in green). Themerging of plugs containing Co²⁺ ions and plugs containing the reactionmixture results in a rapid autocatalytic reaction. By using anamplification cascade in which larger and larger plugs of the reactionmixture are used for amplification, each Co²⁺ ion in a plug can beamplified to about 1010 Co²⁺ ions per plug. The result of amplificationis visually detectable.

The (10 μm)³ plugs are preferably merged with larger (100 μm)³ plugs inthe third channel to give approximately 4×10⁻⁸ mole/L solution of Co²⁺ions. Autocatalytic decomposition in the approximately 10⁻⁹ L plugs willproduce plugs 3809 with about 2.4×10¹⁰ Co²⁺ ions (4×10⁻⁵ mole/L). Theflow rates in this system are preferably controlled carefully to controlthe time that plugs spend in each branch. The time provided foramplification is preferably long enough to allow amplification tosubstantially reach completion, but short enough to prevent or minimizeslow decomposition.

Using different plug sizes is advantageous when merging plugs. Plugswith a size of about (1 μm)³ are preferably formed by flowing a samplecontaining about 3×10⁻⁹ mole/L Co²⁺ through channel 3806. This reactioncan be used to detect Co²⁺ at this, or lower, concentration (Endo etal., “Kinetic determination of trace cobalt(II) by visual autocatalyticindication,” Talanta, 1998, vol. 47, pp. 349-353; Endo et al.,“Autocatalytic decomposition of cobalt complexes as an indicator systemfor the determination of trace amounts of cobalt and effectors,”Analyst, 1996, vol. 121, pp. 391-394.). These plugs have a correspondingvolume of about 10⁻¹⁵ L and carry just a few cobalt ions, on averageabout 1.8 ions per plug (corresponding to a Poisson distribution). Theseplugs 3810 are preferably merged with the (1 μm)³ plugs 3811 containingthe Co(III)-5-Br-PAPS/peroxomonosulfate mixture (about 4×10⁻⁵ mole/L).

Upon autocatalytic decomposition of the complex, the number of Co²⁺ ionsin the merged plug 3807 will increase by a factor of between about 104to 1.2×104 Co²⁺ ions (2×10⁻⁵ mole/L in 2 μm³). These plugs 3807 arepreferably merged with the (10 μm)³ plugs 3811 containing the unstablemixture (about 4×10⁻⁵ mole/L). The concentration of Co²⁺ ions in theseapproximately 10⁻¹² L plugs is preferably about 2×10⁻⁸ mole/L, which issufficient to induce autocatalytic decomposition. The number of Co²⁺ions will increase by a factor of between about 10³ to about 2.4×10⁷ions/plug in plugs 3808. The starting solution is dark violet (ε=9.8×10⁴L mol⁻¹ cm⁻¹ for Co(III)-5-Br-PAPS). Channels are preferably designed tocreate an optical path through at least ten consecutive 100 μm plugs.These plugs will provide an approximately 1-mm long optical path, withabsorbance of the starting 4×10⁻⁵ mole/L solution of about 0.4. Thisabsorbance can be detected by an on-chip photodetector or with the nakedeye. If Co²⁺ is present in the sample solution, an autocatalytic cascadewill result in the disappearance of the color of the reaction mixture.

At low concentrations of Co²⁺ in the sample, the system may showstochastic behavior, that is, not every Co²⁺ ion would give rise to adecomposition cascade. However, the attractive feature of this system isthat thousands of tests can be carried out in a matter of seconds, andstatistics and averaging can be performed. Preferably, a sequence ofcontrolled autocatalytic amplification reactions leads to a visualdetection of single ions.

Example 10—Enzyme Kinetics

A microfluidic chip according to the invention was used to measuremillisecond single-turnover kinetics of ribonuclease A (RNase A; EC3.1.27.5), a well-studied enzyme. Sub-microliter sample consumptionmakes the microfluidic chip especially attractive for performing suchmeasurements because they require high concentrations of both the enzymeand the substrate, with the enzyme used in large excess.

The kinetic measurements were performed by monitoring the steady-statefluorescence arising from the cleavage of a fluorogenic substrate byRNase A as the reaction mixture flowed down the channel (see FIG.40(a)). In FIG. 40, a substrate, buffer, and RNase A were introducedinto inlets 401, 401, and 403, respectively. A carrier fluid flowedthrough channel 404. The amount of the product at a given reaction timet [s] was calculated from the intensity of fluorescence at thecorresponding distance point d [m] (t=d/U where U=0.43 m/s is thevelocity of the flow). The channels were designed to wind so that rapidchaotic mixing was induced, and were designed to fit within the field ofview of the microscope so that the entire reaction profile could bemeasured in one spatially resolved image. Selwyn's test (Duggleby, R.G., Enzyme Kinetics and Mechanisms, Pt D; Academic Press: San Diego,1995, vol. 249, pp. 61-90; Selwyn, M. J. Biochim. Biophys. Acta, 1965,vol. 105, pp. 193-195) was successfully performed in this system toestablish that there were no factors leading to product inhibition orRNase A denaturation.

The flow rate of the stock solution of 150 μM of RNase A was keptconstant to maintain 50 μM of RNase A within the plugs. By varying theflow rates of the buffer and substrate (see FIG. 45), progress curveswere obtained for eight different substrate concentrations. For[E]o>>[S]o, the simple reaction equation is [P]t=[S]o(1−Exp(−kt)), where[E]o is the initial enzyme concentration, [S]o is the initial substrateconcentration, [P]t is the time-dependent product concentration and k[s⁻¹] is the single-turnover rate constant. To obtain a more accuratefit to the data, the time delay Δtn required to mix a fraction of thereaction mixture f_(n) was accounted for.

An attractive feature of the microfluidic system used is that thereaction mixture can be observed at time t=0 (there is no dead-time).This feature was used to determine Δtn and fn in this device byobtaining a mixing curve using fluo-4/Ca²⁺ system as previouslydescribed (Song et al., Angew. Chem. Int. Ed. 2002, vol. 42, pp.768-772), and correcting for differences in diffusion constants (Stroocket al., Science, 2002, vol. 295, pp. 647-651). All eight progress curvesgave a good fit with the same rate constant of 1100±250 s⁻¹. The simplertheoretical fits gave indistinguishable rate constants. These resultsare in agreement with previous studies, where cleavage rates ofoligonucleotides by ribonucleases were shown to be ˜10³ s⁻¹.

Thus, this example demonstrates that millisecond kinetics withmillisecond resolution can be performed rapidly and economically using amicrochannel chip according to the invention. Each fluorescence imagewas acquired for 2 s, and required less than 70 nL of the reagentsolutions. These experiments with stopped-flow would require at leastseveral hundreds of microliters of solutions. Volumes of about 2 μL aresufficient for ˜25 kinetic experiments over a range of concentrations.Fabrication of these devices in PDMS is straightforward (McDonald, etal., Accounts

[P]_(t) =Σf _(n)[S]₀(1−Exp(−k(t−Δt _(n))))

Chem. Res. 2002, vol. 35, pp. 491-499) and no specialized equipmentexcept for a standard microscope with a CCD camera is needed to run theexperiments. This system could serve as an inexpensive and economicalcomplement to stopped-flow methods for a broad range of kineticexperiments in chemistry and biochemistry.

Example 11—Kinetics of RNA Folding

The systems and methods of the present invention are preferably used toconduct kinetic measurements of, for example, folding in the time rangefrom tens of microseconds to hundreds of seconds. The systems andmethods according to the invention allow kinetic measurements using onlysmall amounts of sample so that the folding of hundreds of different RNAmutants can be measured and the effect of mutation on foldingestablished. In one aspect according to the invention, the kinetics ofRNA folding is preferably measured by adding Mg²⁺ to solutions ofpreviously synthesized unfolded RNA labeled with FRET pairs in differentpositions. In accordance with the invention, the concentrations of Mg²⁺are preferably varied in the 0.04 to 0.4 μM range by varying the flowrates (see, for example, FIGS. 25a )-c)) to rapidly determine thefolding kinetics over a range of conditions. The ability to integratethe signal over many seconds using the steady-flow microfluidic devicesaccording to the invention can further improve sensitivity.

As shown in FIGS. 25a )-c), the concentrations of aqueous solutionsinside the plugs can be controlled by changing the flow rates of theaqueous streams. In FIGS. 25a )-c), aqueous streams were introduced intoinlets 251-258 wherein flow rates of about 0.6 μL/min for the twoaqueous streams and 2.7 μL/min was used for the third stream. The streamwith the 2.7 μL/min volumetric flow rate was introduced in the left,middle, and right inlet in FIGS. 25a )-c), respectively. A carrier fluidin the form of perfluorodecaline was introduced into channel 259, 260,261. The corresponding photographs on each of the right side of FIGS.25a )-c) illustrate the formation of plugs with different concentrationsof the aqueous streams. The various shadings inside the streams andplugs arise from the use of aqueous solutions of food dyes (red/dark andgreen/light), which allowed visualization, and water were used as thethree streams, the darker shading arising mainly from the red dye colorwhile the lighter shading arising mainly from the green dye color. Thedark stream is more viscous than the light stream, therefore it movesslower (in mm/s) and occupies a larger fraction of the channel at agiven volumetric flow rate (in μL/min).

Example 12—Nanoparticle Experiments with and without Plugs

FIG. 15 illustrates a technique for the synthesis of CdS nanoparticles155. In one experiment, nanoparticles were formed in a microfluidicnetwork. The channels of the microfluidic device had 50 μm×50 μmcross-sections. A fluorinated carrier-fluid (10:1 v/v mixture ofperfluorohexane and 1H,1H,2H,2H-perfluorooctanol) was flowed through themain channel at 15 μm min⁻¹. An aqueous solution, pH=11.4, of 0.80 mMCdCl₂ and 0.80 mM 3-mercaptopropionic acid was flowed through theleft-most inlet channel 151 at 8 μL min⁻¹. An aqueous solution of 0.80mM polyphosphates Na(PO₃)_(n) was flowed through the central inletchannel 152 at 8 μL min⁻¹, and an aqueous solution of 0.96 mM Na₂S wasflowed through the right-most inlet channel 153 at 8 μL min⁻¹. Toterminate the growth of nanoparticles, an aqueous solution of 26.2 mM3-mercaptopropionic acid, pH=12.1, was flowed through the bottom inletof the device 157 at 24 μM min⁻¹. FIG. 15 shows various regions orpoints along the channel corresponding to regions or points wherenucleation 154, growth 158, and termination 156 occurs. Based on theUV-VIS spectrum, substantially monodisperse nanoparticles formed in thisexperiment.

Nanoparticles were also formed without microfluidics. Solutions ofCdCl₂, polyphosphates, Na₂S, and 3-mercaptopropionic acid, identical tothose used in the microfluidics experiment, were used. 0.5 mL of thesolution of CdCl₂ and 3-mercaptopropionic acid, 0.5 mL of polyphosphatessolution, and 0.5 mL of Na₂S solution were combined in a cuvette, andthe cuvette was shaken by hand. Immediately after mixing, 1.5 mL of 26.2mM 3-mercaptopropionic acid was added to the reaction mixture toterminate the reaction, and the cuvette was again shaken by hand. Basedon the UV-VIS spectrum, substantially polydisperse nanoparticles formedin this experiment.

Example 13

Networks of microchannels were fabricated using rapid prototyping inpolydimethylsiloxane (PDMS). The PDMS was purchased from Dow CorningSylgard Brand 184 Silicone Elastomer. The PDMS devices were sealed afterplasma oxidation treatment in Plasma Prep II (SPI Supplies). The deviceswere rendered hydrophobic by baking the devices at 120° C. for 2-4hours. Microphotographs were taken with a Leica MZ12.5 stereomicroscopeand a SPOT Insight color digital camera (Model #3.2.0, DiagnosticInstruments, Inc.). Lighting was provided from a Machine Vision StrobeX-strobe X1200 (20 Hz, 12 μF, 600V, Perkin Elmer Optoelectronics). Toobtain an image, the shutter of the camera was opened for 1 second andthe strobe light was flashed once with the duration of approximately 10μs.

Aqueous solutions were pumped using 10 μl or 50 μl Hamilton Gastightsyringes (1700 series). Carrier-fluid was pumped using 50 μl HamiltonGastight syringes (1700 series). The syringes were attached tomicrofluidic devices by means of Teflon tubing (Weico Wire & Cable Inc.,30 gauge). Syringe pumps from Harvard Apparatus (PHD 2000) were used toinject the liquids into microchannels.

Microbatch crystallization conditions can be achieved. This experimentshows that size of plugs can be maintained and evaporation of waterprevented. In this case, the PDMS device has been soaked in waterovernight before the experiment in order to saturate PDMS with water.The device was kept under water during the experiment. During theexperiment, the flow rates of carrier-fluid and NaCl solution were 2.7μL/min and 1.0 μL/min, respectively. The flow was stopped by cutting offthe Teflon tubing of both carrier-fluid and NaCl solution.

FIG. 16 shows a schematic illustration of a microfluidic deviceaccording to the invention and a microphotograph of plugs of 1M aqueousNaCl sustained in oil. The carrier-fluid is perfluorodecaline with 2%1H,1H,2H,2H-perfluorooctanol. Inside a microchannel, plugs showed noappreciable change in size.

B. Vapor Diffusion Crystallization in Microchannels: ControllingEvaporation of Water from Plugs

This experiment shows that evaporation of water from plugs can becontrolled by soaking devices in water for shorter amounts of time ornot soaking at all. The rate of evaporation can be also controlled bythe thickness of PDMS used in the fabrication of the device. Evaporationrate can be increased by keeping the device in a solution of salt orother substances instead of keeping the device in pure water.

The plug traps are separated by narrow regions that help force the plugsinto the traps. In this experiment, a composite glass/PDMS device wasused. PDMS layer had microchannel and a microscopy slide (Fisher,35×50-1) was used as the substrate. Both the glass slide and the PDMSwere treated in plasma cleaner (Harrick) then sealed. The device wasmade hydrophobic by first baking the device at 120° C. for 2-4 hoursthen silanizing it by(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UnitedChemical Technologies, Inc.).

During the experiment, a flow of carrier-fluid at 1.0 μL/min wasestablished, then flow of aqueous solution was established at a totalrate of 0.9 μL/min. Plug formation was observed inside the microchannel.The flow was stopped approximately 5-10 minutes afterwards by applying apressure from the outlet and stopping the syringe pumps at the sametime.

FIGS. 41(b)-41(c) show a microphotograph (middle and right side) of thewater plugs region of the microfluidic network. FIGS. 41(b)-(c) show theplugs at time t=0 and t=2 hours, respectively. Red aqueous solution is50% waterman red ink in 0.5 M NaCl solution. Ink streams were thenintroduced into inlets 411, 412, 413. An oil stream flowed throughchannel 414. The carrier-fluid is FC-3283 (3M Fluorinert Liquid) with 2%1H,1H,2H,2H-perfluorodecanol. This photograph demonstrates that theevaporation of water through PDMS can be controlled, and thus theconcentration of the contents inside the drops can be increased (this isequivalent to microbatch crystallization). FIG. 41(a) shows a diagram ofthe microfluidic network.

C. Controlling Shape and Attachment of Water Plugs

During the experiment, a flow of carrier fluid at 1.0 μL/min wasestablished, then flow of aqueous solution was established at a totalrate of 2.1 μL/min. Plug formation was observed inside the microchannel.The flow was stopped approximately 5-10 minutes afterwards by applying apressure from the outlet and stopping the syringe pumps at the sametime.

FIG. 39 shows a diagram (left side) of a microfluidic network accordingto the invention. Aqueous streams were introduced into inlets 3901,3902, 3903 while an oil stream flowed through channel 3904. FIG. 39 alsoshows a microphotograph (right side) of the water plug region of themicrofluidic network. This image shows water plugs attached to the PDMSwall. This attachment occurs when low concentrations of surfactant, orless-effective surfactants are used. In this case1H,1H,2H,2H-perfluorooctanol is less effective than1H,1H,2H,2H-perfluorodecanol. In this experiment the oil is FC-3283 (3MFluorinert Liquid) with 2% 1H,1H,2H,2H-perfluorooctanol as thesurfactant.

D. Examples of Protein Crystallization

During the experiment, a flow of oil at 1.0 μL/min was established. Thenthe flow of water was established at 0.1 μL/min. Finally flows oflysozyme and precipitant were established at 0.2 μL/min. Plug formationwas observed inside the microchannel. The flow of water was reduced tozero after the flow inside the channel became stable. The flow wasstopped approximately 5-10 minutes afterwards by applying a pressurefrom the outlet and stopping the syringe pumps at the same time.

FIG. 36 depicts lysozyme crystals grown in water plugs in the wells ofthe microfluidic channel. Lysozyme crystals started to appear insideaqueous plugs both inside and outside plug traps in approximately 10minutes. The image of the three crystals in FIG. 36 was taken 1 hourafter the flow was stopped. Lysozyme crystals appear colored becausethey were observed under polarized light. This is common for proteincrystals.

The left side of FIG. 36 is a diagram of a microfluidic networkaccording to the invention while the right side is microphotograph ofthe crystals formed in plugs in the microfluidic network. A precipitant,lysozyme, and water were introduced into inlets 3601, 3602, and 3603,respectively. Oil was flowed through channel 3604. The lysozyme solutioncontains 100 mg/ml lysozyme in 0.05 M sodium acetate (pH 4.7); theprecipitant solution contains 30% w/v PEG (M.W. 5000), 1.0 M NaCl and0.05 M sodium acetate (pH 4.7); The carrier-fluid is FC-3283 (3MFluorinert Liquid) with 10% 1H,1H,2H,2H-perfluoro-octanol. Themicrochannel device was soaked in FC-3283/H2O for one hour beforeexperiment.

FIG. 32 shows that plug traps are not required for formation of crystalsin a microfluidic network. FIG. 32 shows a diagram (left side) of themicrofluidic network. A precipitant was introduced into inlet 321,lysozyme was introduced into inlet 322, and an aqueous stream wasintroduced into inlet 323. Oil was flowed through channel 324. FIG. 32also shows microphotographs (middle and right side) of lysozyme crystalsgrown inside the microfluidic channel. The experimental condition issame as in FIG. 36.

Example 14—Oil-Soluble Surfactants for Charged Surfaces

In accordance with the invention, neutral surfactants that are solublein perfluorinated phases are preferably used to create positively andnegatively-charged interfaces. To create charged surfaces, neutralsurfactants that can be charged by interactions with water, e.g., byprotonation of an amine or a guanidinium group (FIG. 24B), ordeprotonation of a carboxylic acid group (FIG. 24C), are preferablyused. Preferably, charged surfaces are used to repel, immobilize, orstabilize charged biomolecules. Negatively charged surfaces are usefulfor handling DNA and RNA without surface adsorption. Preferably, bothnegatively and positively-charged surfaces are used to control thenucleation of protein crystals. Many neutral fluorinated surfactantswith acidic and basic groups (RfC(O)OH, Rf(CH₂)₂NH₂, Rf(CH₂)₂C(NH)NH₂)are available commercially (Lancaster, Fluorochem, Aldrich).

To synthesize oligoethylene-glycol terminated surfactants, amodification and improvement of a procedure based on the synthesis ofperfluoro non-ionic surfactants is preferably used. In one aspect, thesynthesis relies on the higher acidity of the fluorinated alcohol toprevent the polycondensation of the oligoethylene glycol. The modifiedsynthesis uses a selective benzylation of one of the alcohol groups ofoligoethylene glycol, followed by activation of the other alcohol groupas a tosylate. A Williamson condensation is then performed under phasetransfer conditions followed by a final deprotection step via catalytichydrogenation using palladium on charcoal.

Example 15—Formation of Plugs in the Presence of Fluorinated Surfactantsand Surface Tension

The surface tension of the oil/water interface has to be sufficientlyhigh in order to maintain a low value of capillary number, C.n. Thefluorosurfactant/water interfaces for water-insoluble fluorosurfactantshave not been characterized, but these surfactants are predicted toreduce surface tension similar to that observed in a system involvingSpan on hexane/water interface (about 20 mN/m). The surface tensions ofthe aqueous/fluorous interfaces are preferably measured in the presenceof fluorosurfactants using the hanging drop method. A video microscopyapparatus specifically constructed for performing these measurements hasbeen used to successfully characterize interfaces. FIGS. 24(a)-24(c)illustrate the synthesis of fluorinated surfactants containingperfluoroalkyl chains and an oligoethylene glycol head group.

Example 16—Forming Gradients by Varying Flow Rates

FIG. 42 shows an experiment involving the formation of gradients byvarying the flow rates. In this experiment, networks of microchannelswere fabricated using rapid prototyping in polydimethylsiloxane (PDMS).The width and height of the channel were both 50 μm. 10%1H,1H,2H,2H-perfluorodecanol in perfluoroperhydrophenanthrene was usedas oil. Red aqueous solution prepared from 50% Waterman red ink in 0.5 MNaCl solution was introduced into inlet 421. The oil flowed throughchannel 424 at 0.5 μl/min. Aqueous streams were introduced into inlets422, 423. To generate the gradient of ink in the channel, the totalwater flow rate was gradually increased from 0.03 μl/min to 0.23 μl/minin 20 seconds at a ramp rate of 0.01 μl/min per second. At the sametime, ink flow rate was gradually decreased from 0.25 μl/min to 0.05μl/min in 20 seconds at a ramp rate of −0.01 μl/min per second. Thetotal flow rate was constant at 0.28 μl/min. The established gradient ofink concentration inside the plugs can be clearly seen from FIG. 42: theplugs further from the inlet are darker since they were formed at ahigher ink flow rate.

Example 17—Lysozome Crystallization Using Gradients

FIG. 43 illustrates an experiment involving the formation of lysozomecrystals using gradients. The channel regions 435, 437 correspond tochannel regions with very low precipitant concentration while channelregion 436 corresponds to optimal range of precipitant concentration. Inthis experiment, networks of microchannels were fabricated using rapidprototyping in polydimethylsiloxane (PDMS). The width of the channel was150 μm and the height was 100 μm. 10% 1H,1H,2H,2H-perfluorodecanol inperfluoroperhydrophenanthrene was used as oil.

During the experiment, a flow of oil through channel 434 at 1.0 μl/minwas established. Then the flow of water introduced through inlet 432 wasestablished at 0.2 μmin. The flows of lysozyme introduced through inlet431 and precipitant introduced through inlet 433 were established at 0.2μmin. Plugs formed inside the channel. To create the gradient, waterflow rate was first gradually decreased from 0.35 μmin to 0.05 μl/minover 45 seconds at a ramp rate of (−0.01 μmin per 1.5 seconds), thenincreased back to 0.35 μmin in 45 seconds at a ramp rate of (0.01 μminper 1.5 seconds). At the same time, precipitant flow rate was graduallyincreased from 0.05 μmin to 0.35 μmin in 45 seconds at a ramp rate of(0.01 μl/min per 1.5 seconds), then decreased to 0.05 μmin in 45 secondsat a ramp rate of (−0.01 μl/min per 1.5 seconds). The flow was stoppedby pulling out the inlet tubing immediately after water and precipitantflow rates returned to the starting values. The plugs created in thisway contained constant concentration of the protein. but variableconcentration of the precipitant: the concentration of the precipitantwas lowest in the beginning and the end of the channel, and it peaked inthe middle of the channel (the center row). Only the plugs in the middleof the channel have the optimal concentration of precipitant forlysozyme crystallization, as confirmed by observing lysozyme crystalsinside plugs in the center row. Visualization was performed underpolarized light. Preferably, all flow rates would be varied, not justthe precipitant and water.

1. A method of conducting a reaction within at least one plug within asubstrate comprising the steps of: introducing a carrier-fluid into afirst channel of the substrate; introducing at least two differentplug-fluids which are immiscible with the carrier-fluid into the firstchannel at one or more plug-forming regions; and applying a force to thefirst channel to induce a fluid flow in the substrate to form at leastone plug that comprise a mixture of the plug-fluids; wherein the crosssection of a plug is substantially the same size as the cross section ofthe first channel at the plug-forming region.
 2. The method of claim 1,further comprising the step of accelerating mixing of the plug-fluidswithin a plug by flowing the plugs through a channel geometry designedto implement a baker's transformation.
 3. The method of claim 1, furthercomprising the step of accelerating mixing of the plug-fluids within aplug by varying the composition of at least one of the plug fluids orthe carrier fluid.
 4. The method of claim 1, further comprising the stepof accelerating mixing of the plug-fluids within a plug by varying theviscosity of at least one of the plug-fluids or carrier fluid.
 5. Themethod of claim 1, further comprising the step of accelerating mixing ofthe plug-fluids within a plug by varying the patterns on the channelwalls.
 6. The method of claim 1, further comprising the step ofaccelerating mixing of the plug-fluids within a plug by varying thehydrophilic properties of at least a portion of the first channel or ofa branch channel.
 7. The method of claim 1, further comprising the stepof accelerating mixing of the plug-fluids within a plug by varying theelectric charge of at least a portion of the first channel or a branchchannel.
 8. The method of claim 1, further comprising a step of mergingat least one plug with a second plug downstream of the plug-formingregion in the first channel.
 9. (canceled)
 10. (canceled)
 11. A methodof accelerating the mixing of at least two plug fluids in at least oneplug in a microchannel comprising the steps of: introducing a carrierfluid into a microchannel with at least a straight section and a firstbend, wherein the bend is defined by a radius of curvature from thecenterline of the straight section of the microchannel; forming at leastone plug from at least two plug fluids in the carrier fluid upstream ofthe first bend, wherein the plug fluids do not completely mix at thetime of the forming of the plug, and wherein each of the two plug fluidsare different and immiscible with the carrier fluid; flowing the carrierfluid with the plug through the first bend; wherein the mixing of plugfluids within the plug is accelerated compared to a similarly formedplug flowing through a straight microchannel.
 12. The method of claim11, wherein the microchannel comprises a second bend downstream of thefirst bend.
 13. The method of claim 12, wherein the first bend has adifferent radius of curvature than the second bend.
 14. The method ofclaim 12, wherein the first bend has a similar radius of curvature tothat of the second bend.
 15. The method of claim 11, wherein themicrochannel has a first cross-sectional dimension along a first sectionand a different cross-sectional dimension along a second section. 16.The method of claim 11, wherein the plug fluids do not substantially mixat the time of forming of the plug.
 17. The method of claim 11, whereinthe carrier fluid comprises a fluorinated compound.
 18. A methodcomprising the steps of: introducing a carrier fluid into a microchannelcomprising at least a straight section and a first bend, wherein thebend is defined by a radius of curvature from the centerline of thestraight section of the microchannel; forming at least one plug in thecarrier fluid in the microchannel by introducing a first plug fluidthrough a first inlet located upstream of the first bend and in fluidcommunication with the microchannel so that the plug forms in thecarrier fluid at the junction of the first inlet and the microchannel;introducing a second plug fluid through a second inlet locateddownstream of the first inlet, located upstream of the first bend, andin fluid communication with the microchannel so that the second plugfluid is added to the at least one plug to form a plug containing thefirst and second plug fluids; allowing the plug containing the first andsecond plug fluids to travel through the first bend; wherein the mixingof plug fluids within the plug containing the first and second plugfluids is accelerated compared to a similarly formed plug flowingthrough a straight microchannel; wherein the first and second plugfluids are immiscible with the carrier fluid and are different from eachother.
 19. (canceled)
 20. (canceled)